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Sample records for affinity chromatography revealed

  1. Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein.

    PubMed

    Uhlik, Ondrej; Kamlar, Marek; Kohout, Ladislav; Jezek, Rudolf; Harmatha, Juraj; Macek, Tomas

    2008-12-22

    The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.

  2. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

  3. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  4. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

  5. [Separation of osteoclasts by lectin affinity chromatography].

    PubMed

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  6. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    PubMed

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  7. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  8. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

    2013-12-01

    The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

  9. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  10. Extension of the selection of protein chromatography and the rate model to affinity chromatography.

    PubMed

    Sandoval, G; Shene, C; Andrews, B A; Asenjo, J A

    2010-01-01

    The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

  11. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

  12. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  13. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.

  14. Virus inactivation by protein denaturants used in affinity chromatography.

    PubMed

    Roberts, Peter L; Lloyd, David

    2007-10-01

    Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.

  15. Purification of cytochrome c oxidase by lysine-affinity chromatography.

    PubMed

    Felsch, J; Kotake, S; Copeland, R A

    1992-02-01

    A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.

  16. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    PubMed

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  17. Purification of baculovirus vectors using heparin affinity chromatography

    PubMed Central

    Nasimuzzaman, Md; Lynn, Danielle; van der Loo, Johannes CM; Malik, Punam

    2016-01-01

    Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26–29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications. PMID:27933303

  18. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.

  19. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  20. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  1. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  2. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  3. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  4. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

  5. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  6. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.

  7. Mullerian inhibiting substance fractionation by dye affinity chromatography.

    PubMed

    Budzik, G P; Powell, S M; Kamagata, S; Donahoe, P K

    1983-08-01

    Mullerian inhibiting substance (MIS), a large glycoprotein secreted by the fetal and neonatal testis, is responsible for regression of the Mullerian ducts in the male embryo. This fetal growth regulator has been purified more than 2000-fold from crude testicular incubation medium following fractionation on a triazinyl dye affinity support. A high yield of 60% recovered activity was achieved in the absence of exogenous carrier protein by stabilizing MIS with 2-mercaptoethanol, EDTA, and Nonidet-P40 and eliminating losses in the handling and concentration of MIS fractions. Although affinity elution with nucleotides has proved successful in other systems, MIS could not be eluted with ATP, GTP, or AMP, with or without divalent metal ions. Nucleotide elution, however, does remove contaminating proteins prior to MIS recovery with high ionic strength. The 2000-fold-purified MIS fraction, although not homogeneous, shows a reduction-sensitive band after SDS-gel electrophoresis that has been proposed to be the MIS dimer.

  8. Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

    PubMed

    Fleminger, G; Neufeld, T; Star-Weinstock, M; Litvak, M; Solomon, B

    1992-04-24

    The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.

  9. Mixed-bed affinity chromatography: principles and methods.

    PubMed

    Boschetti, Egisto; Righetti, Pier Giorgio

    2015-01-01

    Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. Other interesting preparative applications of mixed-bed chromatography have since been developed. In this chapter the basic concepts first and then detailed application recipes are described for (1) the reduction of protein dynamic concentration range, (2) the removal of impurity traces at the last stage of a biopurification process, and (3) the selection and use of sorbents as mixed bed in protein purification.

  10. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  11. Using affinity chromatography to engineer and characterize pH-dependent protein switches.

    PubMed

    Sagermann, Martin; Chapleau, Richard R; DeLorimier, Elaine; Lei, Margarida

    2009-01-01

    Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.

  12. Affinity monolith chromatography: A review of principles and recent analytical applications

    PubMed Central

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  13. Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

    PubMed

    London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

    2015-04-01

    Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis.

  14. Glycan-specific whole cell affinity chromatography: a versatile microbial adhesion platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a C-glycoside ketohydrazide affinity chromatography resin that interacts with viable whole-cell microbial populations with biologically appropriate stereo-specificity in a carbohydrate-defined manner. It readily allows for the quantification, selection, and manipulation of target...

  15. Cross-linked leucaena seed gum matrix: an affinity chromatography tool for galactose-specific lectins.

    PubMed

    Seshagirirao, Kottapalli; Leelavathi, Chaganti; Sasidhar, Vemula

    2005-05-31

    A cross-linked leucaena (Leucaena leucocephala) seed gum (CLLSG) matrix was prepared for the isolation of galactose-specific lectins by affinity chromatography. The matrix was evaluated for affinity with a known galactose-specific lectin from the seeds of snake gourd (Trichosanthes anguina). The matrix preparation was simple and inexpensive when compared to commercial galactose-specific matrices (i.e. about 1.5 US dollars/100 ml of matrix). The current method is also useful for the demonstration of the affinity chromatography technique in laboratories. Since leucaena seeds are abundant and inexpensive, and the matrix preparation is easy, CLLSG appears to be a promising tool for the separation of galactose-specific lectins.

  16. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-08

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  17. Conformational kinetics reveals affinities of protein conformational states.

    PubMed

    Daniels, Kyle G; Suo, Yang; Oas, Terrence G

    2015-07-28

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

  18. Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA.

    PubMed

    Sousa, Fani; Prazeres, Duarte M F; Queiroz, João A

    2008-09-01

    The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.

  19. Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes.

    PubMed

    Pimplikar, S W; Reithmeier, R A

    1986-07-25

    Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.

  20. NMR screening of new carbocyanine dyes as ligands for affinity chromatography.

    PubMed

    Cruz, Carla; Boto, Renato E F; Drzazga, Anna K; Almeida, Paulo; Queiroz, João A

    2014-04-01

    Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine-protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography.

  1. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column.

  2. Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand.

    PubMed

    Novotná, Lenka; Hrubý, Martin; Benes, Milan J; Kucerová, Zdenka

    2005-08-19

    Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.

  3. Identification of potential cellular targets of aloisine A by affinity chromatography.

    PubMed

    Corbel, Caroline; Haddoub, Rose; Guiffant, Damien; Lozach, Olivier; Gueyrard, David; Lemoine, Jérôme; Ratin, Morgane; Meijer, Laurent; Bach, Stéphane; Goekjian, Peter

    2009-08-01

    Affinity chromatography was used to identify potential cellular targets of aloisine A (7-n-butyl-6-(4'-hydroxyphenyl)-5H-pyrrolo[2,3b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine.

  4. A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.

    PubMed

    Huang, Yongdong; Zhang, Rongyue; Li, Juan; Li, Qiang; Su, Zhiguo; Ma, Guanghui

    2014-03-01

    Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100-500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.

  5. Preparation of adsorbents for affinity chromatography using TSKgel Tresyl-Toyopearl 650M.

    PubMed

    Nakamura, K; Toyoda, K; Kato, Y; Shimura, K; Kasai, K

    1989-09-08

    The optimum conditions for the coupling of proteins were investigated using TSKgel Tresyl-Toyopearl 650M. They were dependent on the proteins coupled. For example, when soybean trypsin inhibitor was coupled at pH 8 the coupling was completed within 1 h and the subsequent adsorption capacity for trypsin was maximal. Longer coupling times decreased the adsorption capacity due to multi-point attachment. The adsorbents obtained were successfully used for affinity chromatography in a short time.

  6. Optimising the design and operation of semi-continuous affinity chromatography for clinical and commercial manufacture.

    PubMed

    Pollock, James; Bolton, Glen; Coffman, Jon; Ho, Sa V; Bracewell, Daniel G; Farid, Suzanne S

    2013-04-05

    This paper presents an integrated experimental and modelling approach to evaluate the potential of semi-continuous chromatography for the capture of monoclonal antibodies (mAb) in clinical and commercial manufacture. Small-scale single-column experimental breakthrough studies were used to derive design equations for the semi-continuous affinity chromatography system. Verification runs with the semi-continuous 3-column and 4-column periodic counter current (PCC) chromatography system indicated the robustness of the design approach. The product quality profiles and step yields (after wash step optimisation) achieved were comparable to the standard batch process. The experimentally-derived design equations were incorporated into a decisional tool comprising dynamic simulation, process economics and sizing optimisation. The decisional tool was used to evaluate the economic and operational feasibility of whole mAb bioprocesses employing PCC affinity capture chromatography versus standard batch chromatography across a product's lifecycle from clinical to commercial manufacture. The tool predicted that PCC capture chromatography would offer more significant savings in direct costs for early-stage clinical manufacture (proof-of-concept) (∼30%) than for late-stage clinical (∼10-15%) or commercial (∼5%) manufacture. The evaluation also highlighted the potential facility fit issues that could arise with a capture resin (MabSelect) that experiences losses in binding capacity when operated in continuous mode over lengthy commercial campaigns. Consequently, the analysis explored the scenario of adopting the PCC system for clinical manufacture and switching to the standard batch process following product launch. The tool determined the PCC system design required to operate at commercial scale without facility fit issues and with similar costs to the standard batch process whilst pursuing a process change application. A retrofitting analysis established that the direct cost

  7. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling*

    PubMed Central

    Kennedy, Jacob J.; Yan, Ping; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Moore, Heather D.; Lin, Chenwei; Pogosova-Agadjanyan, Era L.; Stirewalt, Derek L.; Reding, Kerryn W.; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  8. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

  9. Polystyrene as an affinity chromatography matrix for the purification of antibodies.

    PubMed

    Staak, C; Salchow, F; Clausen, P H; Luge, E

    1996-08-14

    Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

  10. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-04

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  11. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

    PubMed

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-11-14

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

  12. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

    PubMed

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E; Yates, John R

    2011-11-04

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

  13. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    PubMed

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

  14. Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

    PubMed Central

    Chung, Jinhwa A.; Wollack, James W.; Hovlid, Marisa L.; Okesli, Ayse; Chen, Yan; Mueller, Joachim D.; Distefano, Mark D.; Taton, T. Andrew

    2009-01-01

    Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation. PMID:18834849

  15. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  16. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-15

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

  17. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  18. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  19. Isolation and partial characterization of Bromelia hemisphaerica protease by affinity chromatography.

    PubMed

    Ochoa, N; Agundis, C; Córdoba, F

    1987-01-01

    Hemisphaericin, the protease from Bromelia hemisphaerica fruit juice was isolated by affinity chromatography in one step, using a mercurial sepharose derivative. The enzyme behaves as a single component in immunodifussion, immunoelectrophoresis and polyacrylamide electrophoresis in the presence of SDS and 2-mercaptoethanol. Association and dissociation of active components were evidenced in electrophoresis at pH 3.6 and at pH 8.6. Immunoelectrophoresis analyses also disclosed a certain degree of internal immunological heterogeneity. The results are explained by the presence of an enzyme subunit, of about 8000 daltons, endowed with polymeric properties induced by the pH and oxidative environment.

  20. Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

    PubMed Central

    Berman, Jonathan Dembitz; Young, Michael

    1971-01-01

    Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more. Images PMID:5277092

  1. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

  2. Partial purification of the microsomal rat liver iodothyronine deiodinase. II. Affinity chromatography.

    PubMed

    Mol, J A; van den Berg, T P; Visser, T J

    1988-02-01

    Iodothyronine deiodinase has been solubilized and purified approximately 2400 times from liver microsomal fractions of male Wistar rats pretreated with thyroxine. The deiodinase was solubilized with 1% cholate, and stripped of adhering phospholipids by ammonium sulfate precipitation followed by solubilization with the non-ionic detergent Emulgen 911. The enzyme was further purified by successive ion-exchange chromatography on DEAE-Sephacel and Cellex-P and affinity chromatography on 3,3',5-triiodothyronine-Sepharose. Finally, the deiodinase was reacted with 6-propionyl-2-thiouracil-Sepharose, a derivative of the mechanism-based inhibitor 6-propyl-2-thiouracil. Covalent binding was observed only in the presence of substrate in agreement with the proposed mechanism of deiodination. The deiodinase was eluted from the affinity column by reduction of the enzyme-propylthiouracil mixed disulfide with 50 mM dithiothreitol. The enzyme was approximately 50% pure as judged by SDS-PAGE, exhibiting a subunit molecular weight of 25,000. This preparation was equally enriched in outer ring and inner ring deiodinase activities in keeping with the view that both are intrinsic to a single, type I deiodinase.

  3. A new affinity approach to isolate Escherichia coli 6S RNA with histidine-chromatography.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2010-01-01

    6S RNA is an abundant non-coding RNA in Escherichia coli (E. coli), but its function has not been discovered until recently. The first advance on 6S RNA function was the demonstration of its ability to bind the σ(70)-holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process. The growing interest in the investigation of non-coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA. This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from other E. coli sRNA species. The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt). This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated. A purification strategy was also developed to separate 6S RNA from an sRNA mixture. Pure RNA can be advantageously obtained using the histidine-affinity chromatography method, aiming at its application to structural or functional studies.

  4. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  5. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  6. Application of Frontal Affinity Chromatography to Study the Biomolecular Interactions with Trypsin.

    PubMed

    Hu, YuanYuan; Qian, Junqing; Guo, Hui; Jiang, ShengLan; Zhang, Zheng

    2015-07-01

    Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

  7. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  8. Selective isolation of β-glucan from corn pericarp hemicelluloses by affinity chromatography on cellulose column.

    PubMed

    Yoshida, Tomoki; Honda, Yoichi; Tsujimoto, Takashi; Uyama, Hiroshi; Azuma, Jun-ichi

    2014-10-13

    A combination of anion-exchange chromatography and affinity chromatography on a cellulose column was found to be effective for the isolation of β-(1,3;1,4)-glucan (BG) from corn pericarp hemicelluloses (CPHs). CPHs containing 6.6% BG were extracted from corn pericarp with 6M urea-2 wt% NaOH solution and initially fractionated into neutral and acidic parts by anion exchange chromatography to remove acidic arabinoxylan consisting of arabinose (35.6%) and xylose (50.9%). The neutral fraction (yield; 10.1% on the basis of CPHs) consisting of 1.0% arabinose, 10.1% xylose and 80.3% glucose containing 28.4% BG was then applied to a cellulose column of Whatman CF-11. BG could be recovered from the adsorbed fraction on the cellulose column by elution with 2% NaOH in a yield of 2.6% on the basis of CPHs with a purity of 84.7%. The chemical structure of the isolated corn pericarp BG was confirmed by (13)C NMR spectroscopic, methylation and lichenase treatment analyses. The results indicate that the ratios of (1,4)/(1,3) linkage and cellotriosyl/cellotetraosyl segments of the BG were 2.60 and 2.5, respectively.

  9. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  10. Identification of the Cardiac Beta-Adrenergic Receptor Protein: Solubilization and Purification by Affinity Chromatography

    PubMed Central

    Lefkowitz, Robert J.; Haber, Edgar; O'Hara, Donald

    1972-01-01

    A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified β-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 × 105 liters/mol (as compared to two association constants, 107 and 106 liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for β-adrenergic drugs and antagonists. Images PMID:4507606

  11. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  12. Contamination of ribosome inactivating proteins with ribonucleases, separated by affinity chromatography on red sepharose.

    PubMed

    Wang, H X; Ng, T B; Cheng, C H K; Fong, W P

    2003-05-01

    Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

  13. A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

    PubMed

    Hermida, L; Rodríguez, R; Lazo, L; López, C; Márquez, G; Páez, R; Suárez, C; Espinosa, R; García, J; Guzmán, G; Guillén, G

    2002-03-28

    Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

  14. Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions.

    PubMed

    Hirabayashi, J; Arata, Y; Kasai, K

    2000-08-25

    Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.

  15. Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.

    PubMed

    Wiley, J P; Hughes, K A; Kaiser, R J; Kesicki, E A; Lund, K P; Stolowitz, M L

    2001-01-01

    Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

  16. Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography

    PubMed Central

    Kuehne, Christian; Wedepohl, Stefanie; Dernedde, Jens

    2017-01-01

    l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin. PMID:28125045

  17. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  18. Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

    PubMed Central

    Dean, N; Berk, A J

    1987-01-01

    Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay. Images PMID:3697084

  19. Fractionation of the genetic variants of human alpha 1-acid glycoprotein in the native form by chromatography on an immobilized copper(II) affinity adsorbent. Heterogeneity of the separate variants by isoelectrofocusing and by concanavalin A affinity chromatography.

    PubMed

    Hervé, F; Gomas, E; Duché, J C; Tillement, J P

    1993-05-19

    Fractionation of the three main genetic variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.

  20. New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes.

    PubMed

    Melissis, S C; Rigden, D J; Clonis, Y D

    2001-05-11

    Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg

  1. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

    NASA Astrophysics Data System (ADS)

    Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

  2. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  3. Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

    PubMed Central

    Liang, Aiye; Thakkar, Jay N.; Hindle, Michael; Desai, Umesh R.

    2013-01-01

    Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4 m/z, which most probably arise from variably functionalized (β-O4—β-β-linked trimers and/or a β-O4—β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants. PMID:23122400

  4. Purification and characterization of a Cytisus-type Ulex europeus hemagglutinin II by affinity chromatography.

    PubMed

    Konami, Y; Tsuji, T; Matsumoto, I; Osawa, T

    1981-07-01

    Ulex europeus hemagglutinin II [Cytisus-type anti-H(O) hemagglutinin] inhibited most by di-N-acetylchitobiose has been purified by affinity chromatography on a column of chitobiose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. The purified hemagglutinin was homogeneous by ultracentrifugal analysis and gave a single band by electrophoresis on polyacrylamide gel, and had a molecular weight of 105 000 by sedimentation equilibrium and an isoelectric point of pH 6.66. This hemagglutinin was found to be composed of four, apparently identical, subunits of a molecular weight of 25 000 +/- 2 000 by dodecyl sulphate-polyacrylamide gel electrophoresis, and to contain 10.3% carbohydrate in which mannose (3.7%) was the predominant sugar, with smaller amounts of glucose, glucosamine, xylose, fucose and galactose. Amino acid analysis of the purified hemagglutinin II showed a large amount of aspartic acid and serine, but as little as 0.1 mol/100 mol of cystine or methionine could be detected.

  5. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  6. Purification of human immunoglobulin G autoantibodies to tumor necrosis factor using affinity chromatography and magnetic separation.

    PubMed

    Sennikov, S V; Golikova, E A; Kireev, F D; Lopatnikova, J A

    2013-04-30

    Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.

  7. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    PubMed

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  8. Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

    PubMed

    Sengupta, A; Hage, D S

    1999-09-01

    This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.

  9. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

  10. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  11. Hemoglobin Ypsilanti: a high-oxygen-affinity hemoglobin demonstrated by two automated high-pressure liquid chromatography systems.

    PubMed

    Mais, Daniel D; Boxer, Laurence A; Gulbranson, Ronald D; Keren, David F

    2007-11-01

    Hemoglobin (Hb) Ypsilanti is a rare high-oxygen-affinity hemoglobin. Like other high-oxygen-affinity hemoglobins, Hb Ypsilanti manifests as erythrocytosis. Because the migration of many high-oxygen-affinity variants on alkaline and acid gels does not differ from that of HbA, oxygen-hemoglobin dissociation studies are often used to document their presence. Hb Ypsilanti is a notable exception because its electrophoresis pattern on alkaline gel is highly characteristic, exemplifying the phenomenon of hybrid formation in variant hemoglobins. In the past few years, several laboratories have begun to use high-pressure liquid chromatography (HPLC) as a screen for hemoglobinopathies. We demonstrate the elution profile of Hb Ypsilanti on the 2 most widely used HPLC methods.

  12. Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

    PubMed

    Zacharis, Constantinos K; Kalaitzantonakis, Eftichios A; Podgornik, Ales; Theodoridis, Georgios

    2007-03-09

    In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

  13. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high performance affinity chromatography.

    PubMed

    Li, Haiyan; Ge, Jingwen; Guo, Tao; Yang, Shuo; He, Zhonggui; York, Peter; Sun, Lixin; Xu, Xu; Zhang, Jiwen

    2013-08-30

    It is challenging and extremely difficult to measure the kinetics of supramolecular systems with extensive, weak binding (Ka<10(5)M(-1)), and fast dissociation, such as those composed of cyclodextrins and drugs. In this study, a modified peak profiling method based on high performance affinity chromatography (HPAC) was established to determine the dissociation rate constant of cyclodextrin supramolecular systems. The interactions of β-cyclodextrin with acetaminophen and sertraline were used to exemplify the method. The retention times, variances and the plate heights of the peaks for acetaminophen or sertraline, conventional non-retained substance (H2O) on the β-cyclodextrin bonded column and a control column were determined at four flow rates under linear elution conditions. Then, plate heights for the theoretical non-retained substance were estimated by the modified HPAC method, in consideration of the diffusion and stagnant mobile phase mass transfer. As a result, apparent dissociation rate constants of 1.82 (±0.01)s(-1) and 3.55 (±0.37)s(-1) were estimated for acetaminophen and sertraline respectively at pH 6.8 and 25°C with multiple flow rates. Following subtraction of the non-specific binding with the support, dissociation rate constants were estimated as 1.78 (±0.00) and 1.91 (±0.02)s(-1) for acetaminophen and sertraline, respectively. These results for acetaminophen and sertraline were in good agreement with the magnitude of the rate constants for other drugs determined by capillary electrophoresis reported in the literature and the peak fitting method we performed. The method described in this work is thought to be suitable for other supramolecules, with relatively weak, fast and extensive interactions.

  14. Purification and characterization of two types of Cytisus multiflorus hemagglutinin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Tsuji, T; Matsumoto, I; Osawa, T

    1983-10-01

    Two hemagglutinins were separated from extracts of Cytisus multiflorus seeds by successive affinity chromatographies on columns of galactose- and di- N-acetylchitobiose-Sepharose 4B. One was found to be inhibited by di- N-acetylchitobiose or tri- N-acetylchitotriose and shown to possess anti-H(O) activity [Cytisus-type anti-H(O) hemagglutinin designated as Cytisus multiflorus hemagglutinin I]. The other, which was not a blood group-specific hemagglutinin, was inhibited by galactose or lactose (hemagglutinin II). Hemagglutinins I and II were further purified by gel filtration on Sephacryl S-300. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular weights of the purified hemagglutinins I and II were found to be 86000 by sedimentation equilibrium analysis and 80000 by gel filtration. On disc gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, both hemagglutinins gave a single component of a molecular weight of 42000 +/- 2000, suggesting that these hemagglutinins are dimeric proteins of two identical subunits. Hemagglutinins I and II contain 2.7% and 1.5% carbohydrate, respectively, and only very small amounts of cystine and methionine were detected, but they are rich in aspartic acid and serine. Treatment of human O erythrocytes with a purified H-decomposing enzyme (alpha-L-fucosidase from Bacillus fulminans abolished the agglutinability of the cells with hemagglutinin I. This indicates that the L-fucosyl residue is important even for the H-specificity detected by this di-N-acetylchitobiose-specific hemagglutinin I.

  15. An illustration of the clinical relevance of detecting human antimouse antibody interference by affinity chromatography.

    PubMed

    Koper, N P; Massuger, L F; Thomas, C M; Beyer, C; Crooy, M J

    1999-10-01

    Elevated Cancer antigen 125 (CA 125) serum concentrations (up to 221 kU/1) were measured in a 39 year old woman with a positive family history of breast cancer. The serum determinations were performed with the automated Immulite OM-MA chemiluminescent enzyme immunoassay system (Diagnostic Products). Laparoscopic evaluation of the ovaries did not reveal any abnormalities. CA 125 measurements in the same patient using the automated IMx immunoassay system (Abbott) demonstrated normal serum levels. Using a previously reported chromatography procedure IgG type human antimouse antibody activity was found to be present in the serum samples explaining the falsely elevated levels. To prevent this interference the manufacturer modified the assay system by replacing the monoclonal M11 detection antibody with a rabbit polyclonal antibody. Using the modified OM-MA CA 125 assay results were comparable with the IMx values.

  16. Genomic affinities revealed by GISH suggests intergenomic restructuring between parental genomes of the paleopolyploid genus Zea.

    PubMed

    González, Graciela Esther; Poggio, Lidia

    2015-10-01

    The present work compares the molecular affinities, revealed by GISH, with the analysis of meiotic pairing in intra- and interspecific hybrids between species of Zea obtained in previous works. The joint analysis of these data provided evidence about the evolutionary relationships among the species from the paleopolyploid genus Zea (maize and teosintes). GISH and meiotic pairing of intraspecific hybrids revealed high genomic affinity between maize (Zea mays subsp. mays) and both Zea mays subsp. parviglumis and Zea mays subsp. mexicana. On the other hand, when Zea mays subsp. huehuetenanguensis DNA was probed on maize chromosomes, a lower affinity was detected, and the pattern of hybridization suggested intergenomical restructuring between the parental genomes of maize. When DNA from Zea luxurians was used as probe, homogeneous hybridization signals were observed through all maize chromosomes. Lower genomic affinity was observed when DNA from Zea diploperennis was probed on maize chromosomes, especially at knob regions. Maize chromosomes hybridized with Zea perennis DNA showed hybridization signals on four chromosome pairs: two chromosome pairs presented hybridization signal in only one chromosomal arm, whereas four chromosome pairs did not show any hybridization. These results are in agreement with previous GISH studies, which have identified the genomic source of the chromosomes involved in the meiotic configurations of Z. perennis × maize hybrids. These findings allow postulating that maize has a parental genome not shared with Z. perennis, and the existence of intergenomic restructuring between the parental genomes of maize. Moreover, the absence of hybridization signals in all maize knobs indicate that these heterochromatic regions were lost during the Z. perennis genome evolution.

  17. Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography.

    PubMed Central

    Ripoche, J; Al Salihi, A; Rousseaux, J; Fontaine, M

    1984-01-01

    Human complement factor H was prepared in highly purified form from fresh serum by euglobulin precipitation, DEAE-Sephacel chromatography and Sephacryl S-300 gel filtration. This preparation allowed the recovery of 37% of the initial factor H. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that factor H was homogeneous both in reduced and non-reduced media and exhibited a molecular mass of 150 kDa. Charge-shift experiments clearly showed the presence of hydrophobic sites in the factor H molecule. Charge shifts were observed with two detergent systems (Triton/sodium deoxycholate and Triton/cetyltrimethylammonium bromide). Factor H was able to bind to phenyl-Sepharose. This property allowed us to study two populations of factor H. These two populations exhibited the same physicochemical parameters, but revealed differences in their ability to aggregate in low- and iso-ionic-strength media. The molecular basis and biological significance of this heterogeneity are discussed. Images Fig. 3. Fig. 4. Fig. 5. PMID:6235808

  18. G-quadruplex on oligo affinity support (G4-OAS): an easy affinity chromatography-based assay for the screening of G-quadruplex ligands.

    PubMed

    Musumeci, Domenica; Amato, Jussara; Randazzo, Antonio; Novellino, Ettore; Giancola, Concetta; Montesarchio, Daniela; Pagano, Bruno

    2014-05-06

    A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known G-quadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility.

  19. Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection.

    PubMed

    Yari, Sh; Hadizadeh Tasbiti, A; Fateh, A; Karimi, A; Yari, F; Sakhai, F; Ghazanfari, M; Bahrmand, A

    2011-11-01

    Tuberculosis has been declared a global emergency. The mainstay for its control is the rapid and accurate identification of infected individual. Antibodies to A60, one of the macromolecular antigen complexes of mycobacteria were commonly used in the rapid detection of Mycobacterium tuberculosis. The aim of this study was to prepare specific antibodies against A60 for detection of tuberculosis infection. Specific polyclonal antibodies against A60, (A60-Ab) were prepared in rabbits using 2 boosted injections of the antigen (A60). The antibodies were purified and treated with normal oral flora to remove any non-specific and cross-reactive antibodies. These antibodies were conjugated to CNBr-activated Sepharose 4B and used to isolate subunits of A60 with more specificity for M. tuberculosis. A new affinity column was designed to prepare modified (purified) A60 antigen. Purified A60 antigen (PA60-Ag) was used to develop antibody production by Immunoaffinity chromatography. 113 patients with a confirmed diagnosis of pulmonary TB at Pasteur Institute were selected for the study. The specificity of the results was analyzed with TB-rapid test by using PA60-antibodies. TB-rapid test revealed that normal oral flora-absorbed antibodies could lead to more specific results than that of the non-absorbed antibodies. The developed, modified A60 antibodies, (PA60-Ab)-rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. Moreover, PA60-Ag showed only two protein bands of molecular weight 45 and 66kDa in SDS-PAGE while untreated A60 showed multiple bands. Thus, our study helped in the purification of a novel and well characterized A60 antigen and good diagnostic potential for detecting tuberculosis infection.

  20. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  1. Copper(II)-based metal affinity chromatography for the isolation of the anticancer agent bleomycin from Streptomyces verticillus culture.

    PubMed

    Gu, Jiesi; Codd, Rachel

    2012-10-01

    The glycopeptide-based bleomycins are structurally complex natural products produced by Streptomyces verticillus used in combination therapy against testicular and other cancers. Bleomycin has a high affinity towards a range of transition metal ions with the 1:1 Fe(II) complex relevant to its mechanism of action in vivo and the 1:1 Cu(II) complex relevant to its production from culture. The affinity between Cu(II) and bleomycin was the underlying principle for using Cu(II)-based metal affinity chromatography in this work to selectively capture bleomycin from crude S. verticillus culture. A solution of standard bleomycin was retained at a binding capacity of 300 nmol mL(-1) on a 1-mL bed volume of Cu(II)-loaded iminodiacetate (IDA) resin at pH 9 via the formation of the heteroleptic immobilized complex [Cu(IDA)(bleomycin)]. Bleomycin was eluted from the resin at pH 5 as the metal-free ligand under conditions where pK(a) (IDA)affinity chromatography as a green chemistry platform for streamlined access to this high-value therapeutic agent.

  2. Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue.

    PubMed

    Wilson, J E

    1989-01-01

    The mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.

  3. The antigenicity in guinea pigs and monkeys of three mycobacterial polysaccharides purified by affinity chromatography with concanavalin A.

    PubMed

    Daniel, T M

    1975-06-01

    The antigenicity of 3 polysaccharides purified from culture filtrates of Mycobacterim tuberculosis by affinity chromatography using a concanavalin A-agarose absorbent was studied. All 3 purified polysaccharides were found to be potent elicitors of delayed skin test reactions in sensitized guinea pigs and in a tuberculos monkey. This antigenicity could not be attributed to contaminating protein. Small dermal reactions were also observed in control guinea pigs. All 3 polysaccharides reacted with precipitating antibody in guinea pig sera, the antigenic specificity observed with the guinea pig sera differing from that demonstrated with reference goat antiserum. The 3 polysaccharides were also demonstrated to contain hemagglutination antigenic sites.

  4. [Prospects of application of the chitin-binding domains to isolation and purification of recombinant proteins by affinity chromatography: a review].

    PubMed

    Kurek, D V; Lopatin, S A; Varlamov, V P

    2009-01-01

    Properties of substrate-binding domains, some parameters of affinity sorbents, and a number of other special features that were necessary to take into account during creation of chromatographic system for isolation and purification of proteins with incorporated chitin-binding domain were discussed in this review. This method was shown to be successfully used along with metal-chelate affinity chromatography. The metal-chelate affinity chromatography with the use of polyhistidine peptides as affinity labels is successfully applied to isolation, purification, and investigation of recombinant proteins. However, this system had some disadvantages. At present, scientists attracted more and more attention to substrate-binding domains, including those chitin-binding, because they had a number of advantages being used as affinity label.

  5. Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.

    PubMed

    Merkley, Eric D; Anderson, Brian J; Park, Jea; Belchik, Sara M; Shi, Liang; Monroe, Matthew E; Smith, Richard D; Lipton, Mary S

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide-spectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1×10(-4) was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  6. Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

    SciTech Connect

    Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.; Belchik, Sara M.; Shi, Liang; Monroe, Matthew E.; Smith, Richard D.; Lipton, Mary S.

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  7. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  8. Separation and analysis of cis-diol-containing compounds by boronate affinity-assisted micellar electrokinetic chromatography.

    PubMed

    Wang, Heye; Lü, Chenchen; Li, Hengye; Chen, Yang; Zhou, Min; Ouyang, Jian; Liu, Zhen

    2013-10-01

    Cis-diol-containing compounds (CDCCs) are usually highly hydrophilic compounds and are therefore difficult to separate by conventional reversed-phase-based micellar electrokinetic chromatography (MEKC) due to poor selectivity. Here, we report a new method, called boronate affinity-assisted micellar electrokinetic chromatography (BAA-MEKC), to solve this issue. A boronic acid with a hydrophobic alkyl chain was added to the background electrolyte, which acted as a modifier to adjust the selectivity. CDCCs can covalently react with the boronic acid to form negatively charged surfactant-like complexes, which can partition into micelles formed with a cationic surfactant. Thus, CDCCs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase. To verify this method, eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCCs. The effects of experimental conditions on the separation were investigated. Finally, the BAA-MEKC method was applied to the separation and analysis of nucleosides extracted from human urine. BAA-MEKC exhibited better selectivity and improved separation as compared with conventional MEKC and CZE. Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.

  9. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  10. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  11. Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.

    PubMed

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Yamada, Atsushi; Endo, Mika; Koike, Tohru

    2006-10-01

    While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

  12. Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide.

    PubMed

    Sano, Daisuke; Matsuo, Takahiro; Omura, Tatsuo

    2004-06-01

    The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H(2)N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology

  13. [Affinity chromatography and proteomic screening as the effective method for S100A4 new protein targets discovery].

    PubMed

    Koshelev, Iu A

    2014-01-01

    Affinity chromatography followed by a selective binding proteins identification can be using as effective method for a biological impotent interactions discovery. The molecular structure and their surface charge as and conformational regulation possibilities, which change their surface hydrophobic properties, all they should to taken in account during method optimization process. With the same' method we had identify some new S100A4 target proteins such as cytoskeleton proteins Sept2, Sept7, Sept11 and this interaction would can to highlight as S100A4 would regulate cell motility. Even we had identify the transcription cofactor Ddx5 and through such complex formation a S100A4 protein would can to regulate E-cadherin, p21 Waf1/Cip1), Bnip3 gene expression. The same protocol can be using for a target proteins search with another S100 protein family members, because their molecules demonstrate a high homology level in amino aside sequences and 3D structures.

  14. Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose.

    PubMed

    Perła, Joanna; Undas, Anetta; Twardowski, Tomasz; Jakubowski, Hieronim

    2004-08-05

    Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.

  15. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    PubMed Central

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-01-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  16. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally.

  17. pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling.

    PubMed

    Borrok, M Jack; Wu, Yanli; Beyaz, Nurten; Yu, Xiang-Qing; Oganesyan, Vaheh; Dall'Acqua, William F; Tsui, Ping

    2015-02-13

    The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

  18. Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.

    PubMed

    Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N

    2011-05-25

    Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

  19. Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

    2007-04-10

    In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

  20. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  1. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form.

  2. Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

    PubMed

    Teng, S F; Sproule, K; Husain, A; Lowe, C R

    2000-03-31

    A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

  3. Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment.

    PubMed

    Xia, Hui; Murray, Kermit; Soper, Steven; Feng, June

    2012-02-01

    Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

  4. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

  5. Evolved Streptavidin Mutants Reveal Key Role of Loop Residue in High-affinity Binding

    SciTech Connect

    M Magalhaes; C Melo Czekster; R Guan; V Malashkevich; S Almo; M Levy

    2011-12-31

    We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a {approx}10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.

  6. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  7. Computational Model Reveals Limited Correlation between Germinal Center B-Cell Subclone Abundancy and Affinity: Implications for Repertoire Sequencing

    PubMed Central

    Reshetova, Polina; van Schaik, Barbera D. C.; Klarenbeek, Paul L.; Doorenspleet, Marieke E.; Esveldt, Rebecca E. E.; Tak, Paul-Peter; Guikema, Jeroen E. J.; de Vries, Niek; van Kampen, Antoine H. C.

    2017-01-01

    Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population. For these reasons, several groups successfully proceeded or suggested selecting highly abundant subclones from the repertoire to obtain the Ag-specific B cells. Given the nature of affinity maturation one would expect that abundant subclones are of high affinity but since repertoire sequencing only provides information about abundancies, this can only be verified with additional experiments, which are very labor intensive. Moreover, this would also require knowledge of the Ag, which is often not available for clinical samples. Consequently, in general we do not know if the selected highly abundant subclone(s) are also the high(est) affinity subclones. Such knowledge would likely improve the selection of relevant subclones for further characterization and Ag screening. Therefore, to gain insight in the relation between subclone abundancy and affinity, we developed a computational model that simulates affinity maturation in a single GC while tracking individual subclones in terms of abundancy and affinity. We show that the model correctly captures the overall GC dynamics, and that the amount of expansion is qualitatively comparable to expansion observed from B cells isolated from human lymph nodes. Analysis of the fraction of high- and low-affinity subclones among the unexpanded and expanded subclones reveals a limited correlation between

  8. Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.

    PubMed Central

    Srivastava, P N; Farooqui, A A

    1979-01-01

    Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C. Images Fig. 4. PMID:540029

  9. Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

    PubMed

    Baieli, María F; Urtasun, Nicolás; Martinez, María J; Hirsch, Daniela B; Pilosof, Ana M R; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

    2017-01-01

    Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

  10. New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

    PubMed

    Lauer, I; Bonnewitz, B; Meunier, A; Beverini, M

    2000-01-14

    Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also

  11. Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

    PubMed Central

    Callaghan, J M; Toh, B H; Simpson, R J; Baldwin, G S; Gleeson, P A

    1992-01-01

    We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized

  12. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  13. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  14. Comparative study of glycated hemoglobin by ion exchange chromatography and affinity binding nycocard reader in type 2 diabetes mellitus.

    PubMed

    Gautam, N; Dubey, R K; Jayan, A; Nepaune, Y; Padmavathi, P; Chaudhary, S; Jha, S K; Sinha, A K

    2014-12-01

    The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus (DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n = 100) who visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa, Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their fasting (164.46 ± 45.33 mg/dl) and random (187.93 ± 78.02 mg/dl) serum glucose level along with clinical history highly suggestive of type 2 DM. The HbA1c values of (7.8 ± 1.9%) and (8.0 ± 2.2%) were found in DM patients as estimated by those two different methods respectively. The highest frequency was observed in HbA1c > 8.0% indicating maximum cases were under very poor glycemic control. However, there were no significant differences observed in HbA1c value showing both methods are comparable in nature and can be used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c determination, the availability of report at the time of the patient visit can be made easy by using Nycocard Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control, clinical decision-making and changes in treatment regimen.

  15. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  16. Purification of a lectin from M. rubra leaves using immobilized metal ion affinity chromatography and its characterization.

    PubMed

    Sureshkumar, Thavamani; Priya, Sulochana

    2012-12-01

    Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, D-galactosamine and N-acetyl-D-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4-11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe(2+), Ca(2+), Zn(2+), Ni(2+), Mn(2+), Na(+) and K(+), HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3(+) and Al(3+)) and Cu(2+) did not affect the activity. In the presence of Mg(2+) and Hg(2+), only 25 % of HA activity remained.

  17. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography.

    PubMed

    Machado, Gleyce Alves; Oliveira, Heliana Batista de; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-05-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

  18. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

  19. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

    SciTech Connect

    Niture, Suryakant K.; Doneanu, Catalin E.; Velu, Chinavenmeni S.; Bailey, Nathan I.; Srivenugopal, Kalkunte S. . E-mail: Kalkunte.srivenugopal@ttuhsc.edu

    2005-12-02

    Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

  20. Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

    PubMed Central

    Gilbert, H J; Lowe, C R; Drabble, W T

    1979-01-01

    Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. PMID:44191

  1. Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer

    PubMed Central

    Ambrose, Sarah R.; Gordon, Naheema S.; Goldsmith, James C.; Wei, Wenbin; Zeegers, Maurice P.; James, Nicholas D.; Knowles, Margaret A.; Bryan, Richard T.; Ward, Douglas G.

    2015-01-01

    Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers. PMID:28248271

  2. Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1

    PubMed Central

    Bonza, Cristina; Carnelli, Antonella; De Michelis, Maria Ida; Rasi-Caldogno, Franca

    1998-01-01

    The Ca2+-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thaliana chloroplast envelope. PMID:9490776

  3. Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

    NASA Astrophysics Data System (ADS)

    Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

    2016-10-01

    Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

  4. Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.

    PubMed

    Zhuang, Ran; Zhang, Yuan; Zhang, Rui; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2008-05-01

    GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.

  5. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

    PubMed Central

    Machado, Gleyce Alves; de Oliveira, Heliana Batista; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-01-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (Junbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJunbound) and aqueous (AJunbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for Junbound, 92.5% and 93.5% for DJunboundand 82.5% and 82.6% for AJunbound. By immunoblot, the DJunboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJunboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot. PMID:23778661

  6. LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.

    PubMed

    Song, Ehwang; Zhu, Rui; Hammoud, Zane T; Mechref, Yehia

    2014-11-07

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC-MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC-ESI-MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts.

  7. Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution.

    PubMed

    Jain, Niyati; Lin, Hsuan-Chun; Morgan, Christopher E; Harris, Michael E; Tolbert, Blanton S

    2017-02-28

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5'-YAG-3' and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

  8. Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

    PubMed Central

    Jain, Niyati; Lin, Hsuan-Chun; Morgan, Christopher E.; Harris, Michael E.; Tolbert, Blanton S.

    2017-01-01

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo. PMID:28193894

  9. Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

    PubMed

    Regnault, V; Rivat, C; Vallar, L; Geschier, C; Stolz, J F

    1992-12-11

    The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

  10. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  11. Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.

  12. Analysis of Free Drug Fractions in Serum by Ultrafast Affinity Extraction and Two-Dimensional Affinity Chromatography using α1-Acid Glycoprotein Microcolumns

    PubMed Central

    Bi, Cong; Zheng, Xiwei; Hage, David S.

    2016-01-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830 ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20 min when using only 3–10 µL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  13. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    PubMed

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

  14. Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH.

    PubMed

    Li, Xiaobao; Pennington, Justin; Stobaugh, John F; Schöneich, Christian

    2008-01-15

    Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.

  15. Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

    PubMed

    Robichon, Carine; Luo, Jianying; Causey, Thomas B; Benner, Jack S; Samuelson, James C

    2011-07-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

  16. Chromatography

    MedlinePlus

    Chromatography is a way of separating two or more chemical compounds. Chemical compounds are chemicals that are ... of chemical compound. There are different kinds of chromatography. These include gas, high pressure liquid, or ion ...

  17. Isolation of a Trypanosoma cruzi antigen by affinity chromatography with a monoclonal antibody. Preliminary evaluation of its possible applications in serological tests.

    PubMed Central

    Carbonetto, C H; Malchiodi, E L; Chiaramonte, M; Durante de Isola, E; Fossati, C A; Margni, R A

    1990-01-01

    By affinity chromatography with a monoclonal antibody (163B6), obtained in our laboratory, we have isolated a T. cruzi antigen which could be useful for differential diagnosis of Chagas' disease from leishmaniasis. This antigen, a 52-kD protein, reacted with all sera from Chagas' disease patients tested but not with sera from patients with leishmania, in ELISA. The 52-kD antigen is widely distributed in the Trypanosoma genus since the 163B6 monoclonal antibody reacts with T. rangeli and 8 strains and a clone of T. cruzi epimastigotes. Images Fig. 1 Fig. 2 PMID:2119921

  18. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis.

    PubMed

    Scopes, R K

    1984-02-01

    2-Keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) has been isolated from extracts of Zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. The one-step procedure gives an enzyme with specific activity 600 units mg-1. Only 1 out of 47 dyes, Procion Yellow MX-GR, bound the enzyme completely in 20 mM phosphate buffer, pH 6.5. A column of Navy HE-R adsorbent was used first to remove most of the potentially adsorbing proteins.

  19. Preparation and evaluation of a phenylboronate affinity monolith for selective capture of glycoproteins by capillary liquid chromatography.

    PubMed

    Lin, Zi An; Pang, Ji Lei; Lin, Yao; Huang, Hui; Cai, Zong Wei; Zhang, Lan; Chen, Guo Nan

    2011-08-21

    A phenylboronate affinity monolith was prepared and applied to the selective capture of glycoproteins from unfractionated protein mixtures. The monolith was synthesized in a 100 μm i.d capillary by an in situ polymerization procedure using a pre-polymerization mixture consisting of 4-vinylphenylboronic acid (VPBA) as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, diethylene glycol and ethylene glycol as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The prepared monolith was characterized in terms of the morphology, pore property, and recognition property. The selectivity and dynamic binding capacity were evaluated by using standard glycoproteins and nonglycoproteins as model proteins. The chromatographic results demonstrated that the phenylboronate affinity monolith had higher selectivity and binding capacity for glycoprotein than nonglycoprotein. The resulting phenylboronate affinity monolith was used as the sorbent for in-tube solid phase microextraction (in-tube SPME), and the extraction performance of the monolith was assessed by capture of ovalbumin from egg white sample.

  20. Single-molecule peptide-lipid affinity assay reveals interplay between solution structure and partitioning.

    PubMed

    Matin, Tina R; Sigdel, Krishna P; Utjesanovic, Milica; Marsh, Brendan P; Gallazzi, Fabio; Smith, Virginia F; Kosztin, Ioan; King, Gavin M

    2017-03-27

    Interactions between short protein segments and phospholipid bilayers dictate fundamental aspects of cellular activity and have important applications in biotechnology. Yet, a lack of suitable methodology for directly probing these interactions has hindered mechanistic understanding. We developed a precision atomic force microscope (AFM)-based single-molecule force spectroscopy assay and probed partitioning into lipid bilayers by measuring the mechanical force experienced by a peptide. Protein segments were constructed from the peripheral membrane protein SecA, a key ATPase in bacterial secretion. We focused on the first 10 amino-terminal residues of SecA (SecA2-11) which are known to be lipophilic. In addition to the core SecA2-11 sequence, constructs with nearly identical chemical composition but with differing geometry were used: two copies of SecA2-11 linked in series, and two copies in parallel. Lipid bilayer partitioning interactions of peptides with differing structures were distinguished. To model the energetic landscape, a theory of diffusive barrier crossing was extended to incorporate a superposition of potential barriers with variable weights. Analysis revealed two dissociation pathways for the core SecA2-11 sequence with well-separated intrinsic dissociation rates. Molecular dynamics simulations showed that the three peptides had significant conformational differences in solution that correlated well with measured variations in the propensity to partition into the bilayer. The methodology is generalizable and can be applied to other peptide and lipid species.

  1. Chromatography on DEAE ion-exchange and Protein G affinity columns in tandem for the separation and purification of proteins.

    PubMed

    Qi, Y; Yan, Z; Huang, J

    2001-10-30

    A high-performance liquid-chromatographic method based on coupled DEAE anion-exchange and Protein G affinity columns has been developed for the simultaneous separation and purification of immunoglobulin G and albumin from mouse serum. The diluted mouse serum was injected directly into this system, and the proteins were eluted separately from the DEAE and Protein G columns, coupled in series, by the column-switching technique. The advantages of this method are that IgG and albumin can be separated and purified simultaneously, the expensive affinity column is protected from contamination by the impurities in the mouse serum, and it is fast, selective, robust, and reproducible.

  2. Community analysis reveals close affinities between endophytic and endolichenic fungi in mosses and lichens.

    PubMed

    U'ren, Jana M; Lutzoni, François; Miadlikowska, Jolanta; Arnold, A Elizabeth

    2010-08-01

    Endolichenic fungi live in close association with algal photobionts inside asymptomatic lichen thalli and resemble fungal endophytes of plants in terms of taxonomy, diversity, transmission mode, and evolutionary history. This similarity has led to uncertainty regarding the distinctiveness of endolichenic fungi compared with endophytes. Here, we evaluate whether these fungi represent distinct ecological guilds or a single guild of flexible symbiotrophs capable of colonizing plants or lichens indiscriminately. Culturable fungi were sampled exhaustively from replicate sets of phylogenetically diverse plants and lichens in three microsites in a montane forest in southeastern Arizona (USA). Intensive sampling combined with a small spatial scale permitted us to decouple spatial heterogeneity from host association and to sample communities from living leaves, dead leaves, and lichen thalli to statistical completion. Characterization using data from the nuclear ribosomal internal transcribed spacer and partial large subunit (ITS-LSU rDNA) provided a first estimation of host and substrate use for 960 isolates representing five classes and approximately 16 orders, 32 families, and 65 genera of Pezizomycotina. We found that fungal communities differ at a broad taxonomic level as a function of the phylogenetic placement of their plant or lichen hosts. Endolichenic fungal assemblages differed as a function of lichen taxonomy, rather than substrate, growth form, or photobiont. In plants, fungal communities were structured more by plant lineage than by the living vs. senescent status of the leaf. We found no evidence that endolichenic fungi are saprotrophic fungi that have been "entrapped" by lichen thalli. Instead, our study reveals the distinctiveness of endolichenic communities relative to those in living and dead plant tissues, with one notable exception: we identify, for the first time, an ecologically flexible group of symbionts that occurs both as endolichenic fungi and as

  3. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  4. Immobilized fusion protein affinity chromatography combined with HPLC-ESI-Q-TOF-MS/MS for rapid screening of PPARγ ligands from natural products.

    PubMed

    Zhu, Junfeng; Yi, Xiaojiao; Liu, Wenhui; Xu, Yingchun; Chen, Shuqing; Wu, Yongjiang

    2017-04-01

    Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.

  5. Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins).

    PubMed Central

    Haddad, J G; Kowalski, M A; Sanger, J W

    1984-01-01

    The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein. Images Fig. 1. Fig. 3. Fig. 4. PMID:6547042

  6. Synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide), a conjugate useful for affinity chromatography and for testosterone immunoassays.

    PubMed

    Luppa, P; Hauck, S; Schwab, I; Birkmayer, C; Hauptmann, H

    1996-01-01

    We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide) from 17 beta-hydroxyandrost-4-en-3-one (testosterone) via copper-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhexyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatographic separation of the 7 alpha-isomer from the alpha / beta mixture and cleavage of the silyl ether, the alcohol was oxidized to the 6-hexanal side chain and then subjected to reductive amination. The resulting primary amine is easily biotinylated using biotinyl-N-hydroxysuccinimide ester. The overall yield for the epimeric 7 alpha-end product was 30%. The absolute configurations of the epimers were investigated by 1H NMR studies by the nuclear Overhauser effect. We introduced a biotin label to the testosterone molecule at ring position 7 in compliance with Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the functional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testosterone hapten used to elicit antibodies offers the feasibility of applying the testosterone derivative as an optimal immunoadsorbent in affinity chromatography. The 7 alpha-biotinylated testosterone was used to obtain active antitestosterone antibodies from a specific antiserum by affinity chromatography. This was achieved by attaching the biotinylated testosterone to agarose-coupled streptavidin beads. Accordingly, a 3H-testosterone-binding test demonstrated a 20-fold increase in affinity of the purified antibody to the steroid compared to the original antiserum, and a recovery of > 80% could be obtained. The antitestosterone antibody, obtained by that method, is an effective component for use in a competitive immunoassay for testosterone in human sera. An assay configuration is conceivable with the same 7 alpha-biotinylated testosterone employed as

  7. CSAR scoring challenge reveals the need for new concepts in estimating protein-ligand binding affinity.

    PubMed

    Novikov, Fedor N; Zeifman, Alexey A; Stroganov, Oleg V; Stroylov, Viktor S; Kulkov, Val; Chilov, Ghermes G

    2011-09-26

    The dG prediction accuracy by the Lead Finder docking software on the CSAR test set was characterized by R(2)=0.62 and rmsd=1.93 kcal/mol, and the method of preparation of the full-atom structures of the test set did not significantly affect the resulting accuracy of predictions. The primary factors determining the correlation between the predicted and experimental values were the van der Waals interactions and solvation effects. Those two factors alone accounted for R(2)=0.50. The other factors that affected the accuracy of predictions, listed in the order of decreasing importance, were the change of ligand's internal energy upon binding with protein, the electrostatic interactions, and the hydrogen bonds. It appears that those latter factors contributed to the independence of the prediction results from the method of full-atom structure preparation. Then, we turned our attention to the other factors that could potentially improve the scoring function in order to raise the accuracy of the dG prediction. It turned out that the ligand-centric factors, including Mw, cLogP, PSA, etc. or protein-centric factors, such as the functional class of protein, did not improve the prediction accuracy. Following that, we explored if the weak molecular interactions such as X-H...Ar, X-H...Hal, CO...Hal, C-H...X, stacking and π-cationic interactions (where X is N or O), that are generally of interest to the medicinal chemists despite their lack of proper molecular mechanical parametrization, could improve dG prediction. Our analysis revealed that out of these new interactions only CO...Hal is statistically significant for dG predictions using Lead FInder scoring function. Accounting for the CO...Hal interaction resulted in the reduction of the rmsd from 2.19 to 0.69 kcal/mol for the corresponding structures. The other weak interaction factors were not statistically significant and therefore irrelevant to the accuracy of dG prediction. On the basis of our findings from our

  8. Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification.

    PubMed

    Arica, M Yakup; Yilmaz, Meltem; Yalçin, Emine; Bayramoğlu, Gülay

    2004-06-15

    Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.

  9. Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

    PubMed Central

    De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

    2014-01-01

    Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients

  10. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    PubMed

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.

  11. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft.

    PubMed

    Kandra, Lili; Hachem, Maher Abou; Gyémánt, Gyöngyi; Kramhøft, Birte; Svensson, Birte

    2006-09-18

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.

  12. Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography.

    PubMed Central

    Weisbrod, S T

    1982-01-01

    Nucleosomes from actively transcribed genes (active nucleosomes) contain nonhistone proteins HMG 14 and 17 and are preferentially sensitive to digestion by DNAse I. Active nucleosomes isolated by chromatography on an HMG 14 and 17 glass bead affinity column were analyzed with respect to overall structure, accessory nonhistone components and modifications to the DNA and histones. The experiments lead to the following conclusions: the DNA in the active nucleosome is undermethylated compared to bulk DNA; topoisomerase I is a non-stoichiometric component of the active nucleosome fraction; the level of histone acetylation is enriched in active nucleosomes, but the extent of enrichment cannot account for HMG binding; and the two histone H3 molecules in the active nucleosome can dimerize more readily and are, therefore, probably closer together than those in the bulk of the nucleosomes. Additionally it is shown that HMG 14 and 17 prefer to bind to single- vs. double-stranded nucleic acids. The role of HMG 14 and 17 in producing a highly DNAse I sensitive structure and correspondingly helping to facilitate transcription is discussed in terms of these properties. Images PMID:6210882

  13. Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library.

    PubMed

    Smith, Richard G; Missailidis, Sotiris; Price, Michael R

    2002-01-05

    A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.

  14. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.

  15. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    PubMed

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  16. Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector.

    PubMed

    Martínez-Gómez, Maria Amparo; Escuder-Gilabert, Laura; Villanueva-Camañas, Rosa M; Sagrado, Salvador; Medina-Hernández, Maria J

    2008-10-01

    The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n(1) = 0.84; K(1) = 9.7 +/- 0.3x10(3 )M(-1) and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 +/- 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 muM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2-8x10(-4) M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.

  17. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

    PubMed

    Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U

    2016-02-15

    Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.

  18. Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.

    PubMed

    Zhao, Yingxin; Zhang, Wei; White, Michael A; Zhao, Yingming

    2003-08-01

    Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.

  19. Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration.

    PubMed

    Robinson, F Darlene; Moxley, Robert A; Jarrett, Harry W

    2004-01-23

    C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred. After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.

  20. Structure of a TCR with High Affinity for Self-antigen Reveals Basis for Escape from Negative Selection

    SciTech Connect

    Y Yin; Y Li; M Kerzic; R Martin; R Mariuzza

    2011-12-31

    The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3 conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

  1. Paleogenetic Analyses Reveal Unsuspected Phylogenetic Affinities between Mice and the Extinct Malpaisomys insularis, an Endemic Rodent of the Canaries

    PubMed Central

    Gros-Balthazard, Muriel; Hughes, Sandrine; Alcover, Josep Antoni; Hutterer, Rainer; Rando, Juan Carlos; Michaux, Jacques; Hänni, Catherine

    2012-01-01

    Background The lava mouse, Malpaisomys insularis, was endemic to the Eastern Canary islands and became extinct at the beginning of the 14th century when the Europeans reached the archipelago. Studies to determine Malpaisomys' phylogenetic affinities, based on morphological characters, remained inconclusive because morphological changes experienced by this insular rodent make phylogenetic investigations a real challenge. Over 20 years since its first description, Malpaisomys' phylogenetic position remains enigmatic. Methodology/Principal Findings In this study, we resolved this issue using molecular characters. Mitochondrial and nuclear markers were successfully amplified from subfossils of three lava mouse samples. Molecular phylogenetic reconstructions revealed, without any ambiguity, unsuspected relationships between Malpaisomys and extant mice (genus Mus, Murinae). Moreover, through molecular dating we estimated the origin of the Malpaisomys/mouse clade at 6.9 Ma, corresponding to the maximal age at which the archipelago was colonised by the Malpaisomys ancestor via natural rafting. Conclusion/Significance This study reconsiders the derived morphological characters of Malpaisomys in light of this unexpected molecular finding. To reconcile molecular and morphological data, we propose to consider Malpaisomys insularis as an insular lineage of mouse. PMID:22363563

  2. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  3. Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate.

    PubMed

    Chen, C H; Lee, W C

    2001-06-29

    Non-porous particles having an average diameter of 2.1 microm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S-MMA-GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode. The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S-MMA-GMA) particles were slurry packed into a 1.0 cm x 0.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 microg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level.

  4. Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine.

    PubMed

    Yang, Xiaoting; Hu, Yufei; Li, Gongke

    2014-05-16

    Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N'-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17-243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06-0.80μg/L), good linearity (with R(2) between 0.9979 and 0.9993). The recoveries in urine samples were 81.0-105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1-8.2% and 3.7-10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample.

  5. High performance affinity chromatography (HPAC) as a high-throughput screening tool in drug discovery to study drug-plasma protein interactions.

    PubMed

    Vuignier, Karine; Guillarme, Davy; Veuthey, Jean-Luc; Carrupt, Pierre-Alain; Schappler, Julie

    2013-02-23

    Drug-plasma protein binding is an important parameter that, together with other physicochemical properties such as lipophilicity and pK(a), greatly influences drug absorption, distribution, metabolism, and excretion (ADME). Therefore, it is important for pharmaceutical companies to develop a rapid screening assay to examine plasma protein binding during the early stages of the drug discovery process. Human serum albumin (HSA) and α(1)-acid glycoprotein (AGP) are the most important plasma proteins that are capable of binding drugs. In this work, an automated and high-throughput (<3 min/compound) strategy was developed using high performance affinity chromatography (HPAC) with commercial HSA and AGP columns to evaluate drug-plasma protein interactions for drug screening. A generic gradient was used throughout the study to separate drugs that were weakly and tightly bound to HSA and AGP. To accelerate the analysis time, the system was calibrated in a single run by pooling reference compounds without overloading the column. For both HSA and AGP studies, the developed methods were successfully transferred from HPAC-UV to HPAC-MS with single quadrupole MS detection and ammonium acetate, pH 7.0 as a volatile mobile phase. The MS detection enhanced the sensitivity, selectivity, and throughput of the method by pooling unknown compounds. For HSA analyses, the binding percentages obtained using HPAC were well correlated with the binding percentages from the literature. This method was also able to rank compounds based on their affinity for HSA. Concerning the AGP analyses, the quality of the correlation between the binding percentages obtained in HPAC and those from the literature was weaker. However, the method was able to classify compounds into weak, medium, and strong binders and rank compounds based on their affinity for AGP.

  6. Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis.

    PubMed

    Song, Yufeng; Zhang, Hongxiao; Chen, Chen; Wang, Guiping; Zhuang, Kai; Cui, Jin; Shen, Zhenguo

    2014-04-01

    Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834-840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746-2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological

  7. Potential of human serum albumin as chiral selector of basic drugs in affinity electrokinetic chromatography-partial filling technique.

    PubMed

    Martínez-Gómez, Maria A; Villanueva-Camañas, R M; Sagrado, Salvador; Medina-Hernández, Maria J

    2006-11-01

    The enantiomeric resolution of compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer (BGE), protein, and compound solutions), and plug length. In this paper, the possibility of using HSA as chiral selector for enantioseparation of 28 basic drugs using this methodology is studied. The effect of the physicochemical parameters, the structural properties of compounds, and compound-HSA protein binding percentages over their chiral resolution with HSA is outlined. Based on the results obtained, a decision tree is proposed for the "a priori" prediction of the capability of HSA for enantioseparation of basic drugs in AEKC. The results obtained indicated that enantioresolution of basic compounds with HSA depends on the hydrophobicity, polarity, and molar volume of compounds.

  8. Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

    PubMed

    Roy, Ipsita; Jain, Sulakshana; Teotia, Sunita; Gupta, Munishwar Nath

    2004-01-01

    Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

  9. Multivariate optimization approach for chiral resolution of drugs using human serum albumin in affinity electrokinetic chromatography-partial filling technique.

    PubMed

    Martinez-Gomez, Maria A; Villanueva-Camañas, Rosa M; Sagrado, Salvador; Medina-Hernández, Maria J

    2005-11-01

    The enantiomeric resolution of chiral compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer, protein and compound solutions) and protein solution plug length. In this paper multivariate optimization approaches for chiral separation of four basic drugs (alprenolol, oxprenolol, promethazine and propranolol) using HSA as chiral selector in AEKC-partial filling technique are studied. The experimental conditions to achieve maximum resolution are optimized using the Box-Behnken experimental design. Partial least squares and pareto charts are used to analyse the main effects on the resolution. The experimental resolutions observed for all compounds studied in optimum conditions agree with the estimated values based on response surface models. The results obtained show that the range of experimental conditions that provided enantioresolution narrows as hydrophobicity of analytes decreases. This fact can be explained by assuming that hydrophobicity controls the interaction of basic compounds with HSA.

  10. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

    PubMed

    Gianazza, E; Arnaud, P

    1982-01-01

    The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.

  11. Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid.

    PubMed

    Lauder, Robert M; Huckerby, Thomas N; Nieduszynski, Ian A

    2011-10-01

    Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8-9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.

  12. An in depth proteomic analysis based on ProteoMiner, affinity chromatography and nano-HPLC-MS/MS to explain the potential health benefits of bovine colostrum.

    PubMed

    Altomare, Alessandra; Fasoli, Elisa; Colzani, Mara; Parra, Ximena Maria Paredes; Ferrari, Marina; Cilurzo, Francesco; Rumio, Cristiano; Cannizzaro, Luca; Carini, Marina; Righetti, Pier Giorgio; Aldini, Giancarlo

    2016-03-20

    Bovine colostrum (BC), the initial milk secreted by the mammary gland immediately after parturition, is widely used for several health applications. We here propose an off-target method based on proteomic analysis to explain at molecular level the potential health benefits of BC. The method is based on the set-up of an exhaustive protein data bank of bovine colostrum, including the minor protein components, followed by a bioinformatic functional analysis. The proteomic approach based on ProteoMiner technology combined to a highly selective affinity chromatography approach for the immunoglobulins depletion, identified 1786 proteins (medium confidence; 634 when setting high confidence), which were then clustered on the basis of their biological function. Protein networks were then created on the basis of the biological functions or health claims as input. A set of 93 proteins involved in the wound healing process was identified. Such an approach also permits the exploration of novel biological functions of BC by searching in the database the presence of proteins characterized by innovative functions. In conclusion an advanced approach based on an in depth proteomic analysis is reported which permits an explanation of the wound healing effect of bovine colostrum at molecular level and allows the search of novel potential beneficial effects.

  13. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  14. The purification of human enterokinase by affinity chromatography and immunoadsorption. Some observations on its molecular characteristics and comparisons with the pig enzyme.

    PubMed Central

    Grant, D A; Hermon-Taylor, J

    1976-01-01

    A method is described for the purification of human enterokinase from accumulated duodenal fluid by affinity chromatography using p-aminobenzamidine as the ligand. Resolution was greatest when glycylglycine was substituted as the spacer arm. Purification was not a one-step procedure, and some contamination, principally by the alpha-glucosidases, remained. Their removal was completed by immunoadsorption using antisera raised to enterokinase-free material containing these enzymes, prepared as a by-product of the purification procedure. The final preparation had an activity of 4260 nmol of trypsin/min per mg and was free of other enzymic activity tested. Amino acid and sugar analyses of the highly purified enzyme indicated an acidic glycoprotein containing 57% sugar (neutral sugars 47%, amino sugars 10%). The apparent mol.wts. and Stokes radii of human and pig enterokinase were 296 000 and 316 000, and 5.65 and 5.78 nm respectively. Two isoenzymes were identified for human enterokinase and three for the pig enzyme. Human enterokinase demonstrated a resistance to reduction of disulphide linkages and to sodium dodecyl sulphate binding, which may be related to the need for it to retain its integrity in the digestive environment of the upper small intestine. Antisera to highly purified pig and human enterokinases specifically inhibited enterokinase activity. Immuno-inhibition of intestinal aminopeptidase, maltase and glucoamylase by homologous antisera was not observed. Images PLATE 1 PMID:945736

  15. Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Aryal, Uma K; Krochko, Joan E; Ross, Andrew R S

    2012-01-01

    Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.

  16. Serial lectin affinity chromatography with concavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures of prostatic acid phosphatase in human prostate carcinoma.

    PubMed

    Yoshida, K I; Honda, M; Arai, K; Hosoya, Y; Moriguchi, H; Sumi, S; Ueda, Y; Kitahara, S

    1997-08-01

    Differences between human prostate carcinoma (PCA, five cases) and benign prostatic hyperplasia (BPH, five cases) in asparagine-linked (Asn) sugar-chain structure of prostatic acid phosphatase (PAP) were investigated using lectin affinity chromatography with concanavalin A (Con A) and wheat germ agglutinin (WGA). PAP activities were significantly decreased in PCA-derived PAP, while no significant differences between the two PAP preparations were observed in the enzymatic properties (Michaelis-Menten value, optimal pH, thermal stability, and inhibition study). In these PAP preparations, all activities were found only in the fractions which bound strongly to the Con A column and were undetectable in the Con A unbound fractions and in the fractions which bound weakly to the Con A column. The relative amounts of PAP which bound strongly to the Con A column but passed through the WGA column, were significantly greater in BPH-derived PAP than in PCA-derived PAP. In contrast, the relative amounts of PAP which bound strongly to the Con A column and bound to the WGA column, were significantly greater in PCA-derived PAP than in BPH-derived PAP. The findings suggest that Asn-linked sugar-chain structures are altered during oncogenesis in human prostate and also suggest that studies of qualitative differences of sugar-chain structures of PAP might lead to a useful diagnostic tool for PCA.

  17. Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.

    PubMed Central

    Yerima, A; Safi, A; Gastin, I; Michalski, J C; Saunier, M; Gueant, J L

    1996-01-01

    We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

  18. Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking.

    PubMed

    Savane, Tushar S; Kumar, Sanjit; Janakiraman, Vignesh Narasimhan; Kamalanathan, Agamudi S; Vijayalakshmi, Mookambeswaran A

    2016-05-15

    Pseudobiospecific ligand l-histidine is an inexpensive, highly stable, non-toxic ligand explored successfully over the last twenty years for the purification of immunoglobulins in immobilised histidine ligand affinity chromatography. It is of great interest to know the molecular recognition sites of IgG to immobilized l-histidine. Here, we have used an in silico approach to explore the molecular recognition of l-histidine by IgG. We have assessed the feasible binding modes of histidine and its moieties at different sites of IgG and considered only those binding conformations which are exhibited via the imidazole ring NH group or any other OH donating group apart from the ones which are terminally conjugated with the support matrix. We categorised binding site into two categories; category I: inner binding groove and category II: surface binding groove and observed that the hinge region of IgG has most favourable binding pocket for l-histidine and histidyl moieties. Ser and Tyr residues on the hinge region make several significant interactions with l-histidine and histidyl moieties. In case of Fc region of IgG, l-histidine and histidyl moieties closely resemble the binding modes of Protein A, biomimetic ligand 22/8 and B domain of SpA to IgG. In addition to these we have also observed a significant binding site for l-histidine and histidyl moieties at Fab region of IgG.

  19. Enantioseparation of phenotiazines by affinity electrokinetic chromatography using human serum albumin as chiral selector: application to enantiomeric quality control in pharmaceutical formulations.

    PubMed

    Martínez-Gómez, María Amparo; Sagrado, Salvador; Villanueva-Camañas, Rosa María; Medina-Hernández, Maria José

    2007-01-23

    Nowadays, there is a special interest within the pharmaceutical laboratories to develop single enantiomer formulations and consequently a need for analytical methods to determine the enantiomeric purity of drugs. The present paper deals with the enantiomeric separation of promethazine and trimeprazine enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length, is carried out to obtain enantioresolution of promethazine and trimeprazine. The estimated maximum and optimum resolution of trimeprazine and prometazine enantiomers (Rs=1.74 and 2.01, respectively) corresponded to the following experimental conditions: pH 7.5; [HSA] 170 microM and plug length 190 s and pH 7.6; [HSA] 170 microM and plug length 170 s, for trimeprazine and prometazine, respectively. The developed methodologies were applied for the enantiomeric quality control of promethazine and trimeprazine enantiomers in commercially available pharmaceutical formulations. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make it suitable for quality control of the enantiomeric composition of promethazine and trimeprazine in pharmaceutical preparations.

  20. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions.

  1. Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

    PubMed

    Li, Qian; Zhang, Tianlong; Bian, Liujiao

    2016-03-01

    Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties.

  2. Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

    PubMed

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J; Dahlberg, Leif E; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-07-25

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

  3. Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts

    PubMed Central

    Ciesla, L.; Okine, M.; Rosenberg, A.; Dossou, K.S.S.; Toll, L.; Wainer, I.W.; Moaddel, R.

    2016-01-01

    The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs. PMID:26774122

  4. Separation and quantitation of hepatoma-associated gamma-glutamyltransferase by affinity chromatography with Affi-Gel blue and Con A-Sepharose.

    PubMed

    Izumi, M; Taketa, K

    1983-01-01

    Isozymes of serum gamma-glutamyltransferase (GGT) in patients with hepatocellular carcinoma (HCC) and other liver diseases were separated into two groups by double-affinity column chromatography with Affi-Gel blue and Con A-Sepharose, one recovered in the unbound fraction and the other in the bound fraction. Upon electrophoresis with polyacrylamide gradient gel slabs, the unbound fraction gave a GGTI1 band and a faint II1 band and the bound fraction gave a GGT I band and faint bands of GGT I", II' and X, when the original serum contained hepatoma-associated GGT (I1, I" and II') and high-molecular-weight lipid-protein complex, GGT(X). GGT I was present in all cases as a common isozyme. Other lipoprotein-associated GGT isozymes, III-IX, were removed by passing through Affi-Gel blue. GGT activities of unbound fraction in patients with HCC were generally higher than those in patients with non-HCC liver diseases, although the difference was not significant. When the percent of GGT activity of unbound (unbound + bound) was taken, 54% of patients with HCC had a ratio greater than 22%, whereas none of the healthy subjects or patients with other liver diseases gave values greater than this. The present technique may prove to be a useful clinical test for the diagnosis of HCC.

  5. Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry*

    PubMed Central

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J.; Dahlberg, Leif E.; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-01-01

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided. PMID:24917676

  6. Designed synthesis of Graphene @titania @mesoporous silica hybrid material as size-exclusive metal oxide affinity chromatography platform for selective enrichment of endogenous phosphopeptides.

    PubMed

    Yao, Jizong; Sun, Nianrong; Deng, Chunhui; Zhang, Xiangming

    2016-04-01

    In this work, a novel size-exclusive metal oxide affinity chromatography (SE-MOAC) platform was built for phosphoproteome research. The operation for preparing graphene @titania @mesoporous silica nanohybrids (denoted as G@TiO2@mSiO2) was facile and easy to conduct by grafting titania nanoparticles on polydopamine (PD)-covered graphene, following a layer of mesoporous silica was coated on the outermost layer. The G@TiO2@mSiO2 nanohybrids exhibited high sensitivity with a low detection limit of 5 amol/μL (a total amount of 1 fmol) and high selectivity for phosphopeptides at a mass ratio of phosphopeptides to non-phosphopeptides (1:1000). The size-exclusive capability of the nanohybrids were also demonstrated by enriching the phosphopeptides from the mixture of Bovine Serum Albumin (BSA), α-casein, and β-casein digests with a high mass ratio (β-casein digests: α-casein: BSA, 1:500:500), which was attributed to the large surface area and ordered mesoporous channels. In addition, the G@TiO2@mSiO2 nanohybrids were employed to capture the endogenous phosphopeptides from human serum successfully.

  7. Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.

    PubMed

    Weins, Astrid; Schlondorff, Johannes S; Nakamura, Fumihiko; Denker, Bradley M; Hartwig, John H; Stossel, Thomas P; Pollak, Martin R

    2007-10-09

    Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.

  8. Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin

    PubMed Central

    Oh, Jaeho; Edwards, Erin E.; McClatchey, P. Mason; Thomas, Susan N.

    2015-01-01

    ABSTRACT Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell–cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner. PMID:26349809

  9. The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.

    PubMed

    Habicht, K-L; Frazier, C; Singh, N; Shimmo, R; Wainer, I W; Moaddel, R

    2013-01-01

    BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

  10. Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

    PubMed Central

    Jergil, Bengt; Guilford, Hugh; Mosbach, Klaus

    1974-01-01

    1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3′:5′-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N6-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP–Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50–100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N6-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP–Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4374933

  11. Simultaneous speciation of selenoproteins and selenometabolites in plasma and serum by dual size exclusion-affinity chromatography with online isotope dilution inductively coupled plasma mass spectrometry.

    PubMed

    García-Sevillano, M A; García-Barrera, T; Gómez-Ariza, J L

    2014-04-01

    A method for the simultaneous speciation of selenoproteins and selenometabolites in mouse plasma has been developed based on in series two-dimensional size exclusion and affinity high-performance liquid chromatography (2D/SE-AF-HPLC), using two columns of each type, and hyphenation to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-QMS). The method allows the quantitative determination of selenoprotein P (SeP), extracellular glutathione peroxidase (eGPx), selenoalbumin (SeAlb), and selenometabolites in mouse plasma using species-unspecific isotope dilution (SUID). The 2D chromatographic separation is proposed to remove typical spectral interferences in plasma from chloride and bromide on (77)Se ((40)Ar(37)Cl) and (82)Se ((81)Br(1)H). In addition, the approach increases chromatographic resolution allowing the separation of eGPx from Se metabolites of low molecular mass. The method is robust, reliable, and fast with a typical chromatographic runtime less than 20 min. Precision in terms of relative standard deviation (n = 5) is in the order of 4 %, and detection limits are in the range of 0.2 to 1.0 ng Se g(-1). Method accuracy for determination of total protein bound to Se was assessed by analyzing human serum reference material (BCR-637) certified for total Se content, and latterly applied to mouse plasma (Mus musculus). In summary, a reliable speciation method for the analysis of eGPx, selenometabolites, SeP, and SeAlb in plasma/serum samples is proposed for the first time and is applicable to the evaluation of Se status in human in clinical studies and other mammals for environmental or toxicological assessment.

  12. Mycobacterium tuberculosis dihydrofolate reductase reveals two conformational states and a possible low affinity mechanism to antifolate drugs.

    PubMed

    Dias, Marcio Vinicius Bertacine; Tyrakis, Petros; Domingues, Romenia Ramos; Paes Leme, Adriana Franco; Blundell, Tom L

    2014-01-07

    Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.

  13. High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues*

    PubMed Central

    Szabó, András; Héja, Dávid; Szakács, Dávid; Zboray, Katalin; Kékesi, Katalin A.; Radisky, Evette S.; Sahin-Tóth, Miklós; Pál, Gábor

    2011-01-01

    Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2′ (Leu or Asp), and P4′ (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2′ (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4′ (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (KD 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys51, Arg56, and Arg80) on the surface of human CTRC that interact with the P4′ acidic residue of the inhibitor. The acidic preference of CTRC at P4′ is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation. PMID:21515688

  14. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin.

    PubMed

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-03-20

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.

  15. Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

    PubMed

    Shetty, Kartika N; Latha, Vakada Lavanya; Rao, Rameshwaram Nagender; Nadimpalli, Siva Kumar; Suguna, Kaza

    2013-07-01

    Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

  16. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    PubMed

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug.

  17. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  18. Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level.

    PubMed

    Bartlow, Patrick; Uechi, Guy T; Cardamone, John J; Sultana, Tamanna; Fruchtl, McKinzie; Beitle, Robert R; Ataai, Mohammad M

    2011-08-01

    Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.

  19. Functional chromatography reveals three natural products that target the same protein with distinct mechanisms of action

    PubMed Central

    Kang, MinJin; Wu, Tongde; Wijeratne, E. M. Kithsiri; Lau, Eric C.; Mason, Damian J.; Mesa, Celestina; Tillotson, Joseph; Zhang, Donna D.; Gunatilaka, A. A. Leslie; La Clair, James J.

    2014-01-01

    Access to lead compounds with defined molecular targets continues to be a barrier to the translation of natural product resources. As a solution, we have developed a system that uses discreet, recombinant proteins as the vehicles for natural product isolation. Here, we describe the use of this functional chromatographic method to identify natural products that bind to the AAA+ chaperone, p97, a promising cancer target. Application of this method to a panel of fungal and plant extracts identified rheoemodin, 1-hydroxydehydroherbarin and phomapyrrolidone A as distinct p97 modulators. Excitingly, each of these molecules displayed a unique mechanism of p97 modulation. This discovery provides strong support for the application of functional chromatography to the discovery of protein modulators that would likely escape traditional high-throughput or phenotypic screening platforms. PMID:25125376

  20. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells.

    PubMed

    Bhatia, Prateek A; Moaddel, Ruin; Wainer, Irving W

    2010-06-15

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA, using a baculovirus expression system. The resulting CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [(3)H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9(MRP1)) column, etoposide and furosemide on the CMAC(Sf9(MRP2)) column and etoposide and fumitremorgin C on the CMAC(Sf9(BCPR)) column. The binding affinities (K(i) values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [(3)H]-etoposide on the CMAC(Sf9(MRP1)) column to a greater extent than (R)-verapamil and the relative IC(50) values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC(50) values were consistent with previously reported data. The results indicated that the CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system.

  1. In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake in Streptomyces coelicolor

    PubMed Central

    Nothaft, Harald; Parche, Stephan; Kamionka, Annette; Titgemeyer, Fritz

    2003-01-01

    HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent Km of 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor. PMID:12533468

  2. Ultra-fast liquid chromatography with tandem mass spectrometry determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up.

    PubMed

    Yang, Xihui; Hu, Yichen; Kong, Weijun; Chu, Xianfeng; Yang, Meihua; Zhao, Ming; Ouyang, Zhen

    2014-11-01

    A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r(2) ≥ 0.9993), low limit of detection (0.5-0.8 μg/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 μg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices.

  3. Structures of Streptococcus pneumoniae PiaA and Its Complex with Ferrichrome Reveal Insights into the Substrate Binding and Release of High Affinity Iron Transporters

    PubMed Central

    Cheng, Wang; Li, Qiong; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing

    2013-01-01

    Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an α-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome. PMID:23951167

  4. Combined Crystal Structure of a Type I Cohesin: MUTATION AND AFFINITY BINDING STUDIES REVEAL STRUCTURAL DETERMINANTS OF COHESIN-DOCKERIN SPECIFICITIES.

    PubMed

    Cameron, Kate; Weinstein, Jonathan Y; Zhivin, Olga; Bule, Pedro; Fleishman, Sarel J; Alves, Victor D; Gilbert, Harry J; Ferreira, Luís M A; Fontes, Carlos M G A; Bayer, Edward A; Najmudin, Shabir

    2015-06-26

    Cohesin-dockerin interactions orchestrate the assembly of one of nature's most elaborate multienzyme complexes, the cellulosome. Cellulosomes are produced exclusively by anaerobic microbes and mediate highly efficient hydrolysis of plant structural polysaccharides, such as cellulose and hemicellulose. In the canonical model of cellulosome assembly, type I dockerin modules of the enzymes bind to reiterated type I cohesin modules of a primary scaffoldin. Each type I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type II dockerin that anchors the entire complex to the cell surface by binding type II cohesins of anchoring scaffoldins. In Bacteroides cellulosolvens, however, the organization of the cohesin-dockerin types is reversed, whereby type II cohesin-dockerin pairs integrate the enzymes into the primary scaffoldin, and type I modules mediate cellulosome attachment to an anchoring scaffoldin. Here, we report the crystal structure of a type I cohesin from B. cellulosolvens anchoring scaffoldin ScaB to 1.84-Å resolution. The structure resembles other type I cohesins, and the putative dockerin-binding site, centered at β-strands 3, 5, and 6, is likely to be conserved in other B. cellulosolvens type I cohesins. Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment. This general approach may provide valuable structural information of the cohesin-dockerin interface, in lieu of a definitive crystal structure.

  5. Immobilized metal affinity chromatography in open-loop simulated moving bed technology: purification of a heat stable histidine tagged beta-glucosidase.

    PubMed

    Sahoo, Deepti; Andersson, Jonatan; Mattiasson, Bo

    2009-06-01

    Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged beta-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each of columns for load, wash, elution etc within the SMB. Only the wash and elution are operated with columns in sequence. The beta-glucosidase was purified to almost single band purity with a purification factor of 15 and a recovery of 91%. SMB-performance showed reduced buffer consumption, higher purification fold, a better yield and higher productivity.

  6. Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

    PubMed

    Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

    2014-07-18

    This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins.

  7. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  8. Profiling of cis-Diol-containing Nucleosides and Ribosylated Metabolites by Boronate-affinity Organic-silica Hybrid Monolithic Capillary Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Jiang, Han-Peng; Qi, Chu-Bo; Chu, Jie-Mei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-01-01

    RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5′-deoxy-5′-methylthioadensine, N4-acetylcytidine, 1-ribosyl-N-propionylhistamine and N2,N2,7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers. PMID:25585609

  9. Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

    PubMed Central

    Lu, Jinghua; Chu, Jonathan; Zou, Zhongcheng; Hamacher, Nels B.; Rixon, Mark W.; Sun, Peter D.

    2015-01-01

    Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop–glycan interaction resulted in an ∼20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases. PMID:25561553

  10. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2012-01-01

    The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.

  11. Histamine H1 receptor cell membrane chromatography online high-performance liquid chromatography with mass spectrometry method reveals houttuyfonate as an activator of the histamine H1 receptor.

    PubMed

    Guo, Ying; Han, Shengli; Cao, Jingjing; Zhang, Tao; He, Langchong

    2014-11-01

    Allergy is an abnormal reaction of the body to an allergen. Histamine is responsible for many of the acute symptoms of allergic diseases. Many of the allergic and inflammatory actions of histamine are mediated by the histamine H1 receptor. In the present study, we established a two-dimensional histamine H1 receptor/cell membrane chromatography with online high-performance liquid chromatography and mass spectrometry method for screening potential histamine-activating components in a traditional Chinese medicine injection. The specification of the method was validated by screening, separating, and identifying a mixed standard solution of diphenhydramine hydrochloride, gefitinib, tamsulosin, and nitrendipine. The Yujin injection, an example of traditional Chinese medicine injection, was screened and potential allergic components acting on the histamine H1 receptor were identified. A Ca(2+) flux assay showed that houttuyfonate and Yujin injection induced calcium release in a dose-dependent manner. This suggests that houttuyfonate is an activator of the histamine H1 receptor. The mechanism of houttuyfonate activation involves phosphorylation of the inositol-1,4,5-trisphosphate receptor. In conclusion, this two-dimensional method can rapidly detect and enrich target components isolated from the Yujin injection. This indicates that individuals with an overexpression of the histamine H1 receptor should be aware of possible allergic reactions when receiving the Yujin injection.

  12. A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

    PubMed

    Srivastava, Vivek Kumar; Suneetha, Korivi Jyothiraj; Kaur, Rupinder

    2014-10-01

    Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

  13. Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns.

    PubMed

    Tong, Zenghan; Schiel, John E; Papastavros, Efthimia; Ohnmacht, Corey M; Smith, Quentin R; Hage, David S

    2011-04-15

    Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins.

  14. Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin-I for capturing sub-glycoproteomics from breast cancer and disease-free human sera.

    PubMed

    Selvaraju, Subhashini; El Rassi, Ziad

    2012-07-01

    In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed towards the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease-free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.

  15. Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA.

    PubMed

    Wynne, James W; Di Rubbo, Antonio; Shiell, Brian J; Beddome, Gary; Cowled, Christopher; Peck, Grantley R; Huang, Jing; Grimley, Samantha L; Baker, Michelle L; Michalski, Wojtek P

    2013-01-01

    There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

  16. Purification and Characterisation of Immunoglobulins from the Australian Black Flying Fox (Pteropus alecto) Using Anti-Fab Affinity Chromatography Reveals the Low Abundance of IgA

    PubMed Central

    Shiell, Brian J.; Beddome, Gary; Cowled, Christopher; Peck, Grantley R.; Huang, Jing; Grimley, Samantha L.; Baker, Michelle L.; Michalski, Wojtek P.

    2013-01-01

    There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host. PMID:23308125

  17. Electron Affinity Calculations for Thioethers

    NASA Technical Reports Server (NTRS)

    Sulton, Deley L.; Boothe, Michael; Ball, David W.; Morales, Wilfredo

    1997-01-01

    Previous work indicated that polyphenyl thioethers possessed chemical properties, related to their electron affinities, which could allow them to function as vapor phase lubricants (VPL). Indeed, preliminary tribological tests revealed that the thioethers could function as vapor phase lubricants but not over a wide temperature and hertzian pressure range. Increasing the electron affinity of the thioethers may improve their VPL properties over this range. Adding a substituent group to the thioether will alter its electron affinity in many cases. Molecular orbital calculations were undertaken to determine the effect of five different substituent groups on the electron affinity of polyphenyl thioethers. It was found that the NO2, F, and I groups increased the thioethers electron affinity by the greatest amount. Future work will involve the addition of these groups to the thioethers followed by tribological testing to assess their VPL properties.

  18. Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

    PubMed

    Groves, Maria A T; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2014-01-01

    In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn

  19. Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNaV1.7

    PubMed Central

    Thomas-Tran, Rhiannon; Du Bois, J.

    2016-01-01

    Improper function of voltage-gated sodium channels (NaVs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target NaVs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of NaV subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of NaV through a systematic structure–activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human NaV1.7 (hNaV1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective NaV inhibitors. PMID:27162340

  20. Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca(2+)-binding affinity in a C-domain mutant of cardiac troponin C.

    PubMed

    Veltri, Tiago; de Oliveira, Guilherme A P; Bienkiewicz, Ewa A; Palhano, Fernando L; Marques, Mayra de A; Moraes, Adolfo H; Silva, Jerson L; Sorenson, Martha M; Pinto, Jose R

    2017-04-06

    The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca(2+)-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca(2+) than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca(2+)-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

  1. Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

    PubMed

    Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie

    2017-04-01

    Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.

  2. Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.

    PubMed

    Stocker-Majd, Gisela; Hilbrig, Frank; Freitag, Ruth

    2008-06-13

    Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

  3. Proteomic study of serum using gel chromatography and MALDI-TOF MS reveals diagnostic biomarkers in male patients with liver-cancer

    NASA Astrophysics Data System (ADS)

    Zeng, Xin-Hua; Huang, He-Qing; Chen, Dong-Shi; Jin, Hong-Wei; Huang, Hui-Ying

    2007-03-01

    Human serum has been widely employed clinically for diagnosing various fatal diseases. However, the concentration of most proteins in human serum is too low to be directly measured using normal analytical methods. In order to obtain reliable analytical results from proteomic analysis of human serum, appropriate sample preparation is essential. A combined off-line analytical technique of gel chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been successfully established to separate proteins for MS analysis. Using these combined techniques, 176 mass signal peaks of proteins/peptides were found in 6 of 18 fractions from normal male serum (NMS) in the presence of buffer consisting of NH4HCO3 and acetonitrile. A simple gel chromatography column packed with Sephadex G-50 removed most signal-suppressing compounds such as salts and high abundance proteins (HAP). The molecular mass to charge (m/z) ratios of differential peptides revealed in serum of male patient with liver-cancer (LCMPS) compared to NMS were 5365, 5644 and 6462, and these peptides can be used as biomarkers to clinically diagnose liver-cancer. The simple and convenient chromatographic method described here is not only superior to recently described HPLC separation for MS analysis, but also reveals many novel and significant serum biomarkers for the clinical diagnosis of various diseases.

  4. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  5. Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites

    PubMed Central

    López-Gutiérrez, Borja; Dinglasan, Rhoel R.

    2017-01-01

    The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum. The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector. PMID:28104756

  6. Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites.

    PubMed

    López-Gutiérrez, Borja; Dinglasan, Rhoel R; Izquierdo, Luis

    2017-03-07

    The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.

  7. Plant defense responses in opium poppy cell cultures revealed by liquid chromatography-tandem mass spectrometry proteomics.

    PubMed

    Zulak, Katherine G; Khan, Morgan F; Alcantara, Joenel; Schriemer, David C; Facchini, Peter J

    2009-01-01

    Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.

  8. Hydrocarbon Specificity During Aerobic oil Biodegradation Revealed in Marine Microcosms With the use of Comprehensive, Two-Dimensional Gas Chromatography.

    NASA Astrophysics Data System (ADS)

    Wardlaw, G. D.; Reddy, C. M.; Nelson, R. K.; Valentine, D. L.

    2008-12-01

    In 2003 the National Research Council reported more than 380 million gallons of oil is emitted into the ocean each year from natural seepage and as a result of anthropogenic activities. Many of the hydrocarbons making up this oil are persistent and toxic to marine life. Petroleum emitted into biologically sensitive areas can lead to environmental stress and ecosystem collapse. As a result many studies and a substantial amount of resources have been devoted to creating efficient and effective remediation tools and developing a better understanding of natural hydrocarbon weathering processes occurring in marine environments. The goal of this study is to elucidate patterns and extent of aerobic hydrocarbon degradation in marine sediments. In order to assess the specific molecular transformations occurring in petroleum emitted into oxic marine environments, we prepared microcosm experiments using sediments and seawater collected from the natural oil seeps offshore Coal Oil Point, California. Petroleum recovered from Platform Holly in the Santa Barbara Channel, was added to a sediment-seawater mixture and the microcosm bottles were allowed to incubate under aerobic conditions for slightly more than 100 days. Comprehensive, two-dimensional gas chromatography was employed in this study to quantify changes in the concentrations of individual hydrocarbon compounds because of the increased resolution and resolving power provided with this robust analytical method. We show significant hydrocarbon mass loss due to aerobic biodegradation for hundreds of tracked compounds in the microcosm bottles. The results shown here provide quantitative evidence for broad-scale metabolic specificity during aerobic hydrocarbon degradation in surface and shallow subsurface marine sediments.

  9. Virtual Screening of Plant Volatile Compounds Reveals a High Affinity of Hylamorpha elegans (Coleoptera: Scarabaeidae) Odorant-Binding Proteins for Sesquiterpenes From Its Native Host

    PubMed Central

    Palma-Millanao, Rubén; Yáñez, Osvaldo; Rojas, Maximiliano; Mutis, Ana; Venthur, Herbert; Quiroz, Andrés; Ramírez, Claudio C.

    2016-01-01

    Hylamorpha elegans (Burmeister) is a native Chilean scarab beetle considered to be a relevant agricultural pest to pasture and cereal and small fruit crops. Because of their cryptic habits, control with conventional methods is difficult; therefore, alternative and environmentally friendly control strategies are highly desirable. The study of proteins that participate in the recognition of odorants, such as odorant-binding proteins (OBPs), offers interesting opportunities to identify new compounds with the potential to modify pest behavior and computational screening of compounds, which is commonly used in drug discovery, may help to accelerate the discovery of new semiochemicals. Here, we report the discovery of four OBPs in H. elegans as well as six new volatiles released by its native host Nothofagus obliqua (Mirbel). Molecular docking performed between OBPs and new and previously reported volatiles from N. obliqua revealed the best binding energy values for sesquiterpenic compounds. Despite remarkable divergence at the amino acid level, three of the four OBPs evaluated exhibited the best interaction energy for the same ligands. Molecular dynamics investigation reinforced the importance of sesquiterpenes, showing that hydrophobic residues of the OBPs interacted most frequently with the tested ligands, and binding free energy calculations demonstrated van der Waals and hydrophobic interactions to be the most important. Altogether, the results suggest that sesquiterpenes are interesting candidates for in vitro and in vivo assays to assess their potential application in pest management strategies. PMID:27012867

  10. COII/tRNA[sup Lys] intergenic 9-bp deletion and other mtDNA markers clearly reveal that the Tharus (Southern Nepal) have oriental affinities

    SciTech Connect

    Passarino, G.; Semino, O.; Santachiara-Benerecetti, A.S.; Modiano, G. )

    1993-09-01

    The authors searched for the East Asian mtDNA 9-bp deletion in the intergenic COII/tRNA[sup Lys] region in a sample of 107 Tharus (50 from central Terai and 57 from eastern Terai), a population whose anthropological origin has yet to be completely clarified. The deletion, detected by electrophoresis of the PCR-amplified nt 7392-8628 mtDNA fragment after digestion with HaeIII, was found in about 8% of both Tharu groups but was found in none of the 76 Hindus who were examined as a non-Oriental neighboring control population. A complete triplication of the 9-bp unit, the second case so far reported, was also observed in one eastern Tharu. All the mtDNAs with the deletion, and that with the triplication, were further characterized (by PCR amplification of the relevant mTDNA fragments and their digestion with the appropriate enzymes) to locate them in the Ballinger et al. phylogeny of East Asian mtDNA haplotypes. The deletion was found to be associated with four different haplotypes, two of which are reported for the first time. One of the deletions and especially the triplication could be best explained by the assumption of novel length-change events. Ballinger's classification of East Asian mtDNA haplotypes is mainly based on the phenotypes for the DdeI site at nt 10394 and the AluI site at nt 10397. Analysis of the entire Tharu sample revealed that more than 70% of the Tharus have both sites, the association of which has been suggested as an ancient East Asian peculiarity. These results conclusively indicate that the Tharus have a predominantly maternal Oriental ancestry. Moreover, they show at least one and perhaps two further distinct length mutations, and this suggests that the examined region is a hot spot of rearrangements. 21 refs., 5 figs., 6 tabs.

  11. Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

    PubMed Central

    Pawlik, Timothy M; Hawke, David H; Liu, Yanna; Krishnamurthy, Savitri; Fritsche, Herbert; Hunt, Kelly K; Kuerer, Henry M

    2006-01-01

    Background Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. Results 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer. PMID:16542425

  12. Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin

    SciTech Connect

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. )

    1990-04-03

    This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.

  13. On-line monitoring of Soxhlet extraction by chromatography and mass spectrometry to reveal temporal extract profiles.

    PubMed

    Chen, Ssu-Ying; Urban, Pawel L

    2015-06-30

    Soxhlet extraction is a popular sample preparation technique used in chemical analysis. It enables liberation of molecules embedded in complex matrices (for example, plant tissues, foodstuffs). In most protocols, samples are analyzed after the extraction process is complete. However, in order to optimize extraction conditions and enable comparisons between different types of extraction, it would be desirable to monitor it in real time. The main development of this work is the design and construction of the interface between Soxhlet extractor and GC-MS as well as ESI-MS system. The temporal extract profiles, obtained in the course of real-time GC-MS monitoring, have been fitted with mathematical functions to analyze extraction kinetics of different analytes. For example, the mass transfer coefficients of pinene, limonene and terpinene in lemon sample, estimated using the first-order kinetic model, are 0.540h(-1), 0.507h(-1) and 0.722h(-1), respectively. On the other hand, the Peleg model provides the following extraction rates of pinene, limonene and terpinene: 0.370nMh(-1), 0.216nMh(-1) and 0.596nMh(-1), respectively. The results suggest that both first-order kinetic and Peleg equations can be used to describe the progress of Soxhlet extraction. On-line monitoring of Soxhlet extraction reveals extractability of various analytes present in natural samples (plant tissue), and can potentially facilitate optimization of the extraction process.

  14. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  15. Metabolic Profiling with Gas Chromatography-Mass Spectrometry and Capillary Electrophoresis-Mass Spectrometry Reveals the Carbon-Nitrogen Status of Tobacco Leaves Across Different Planting Areas.

    PubMed

    Zhao, Jieyu; Zhao, Yanni; Hu, Chunxiu; Zhao, Chunxia; Zhang, Junjie; Li, Lili; Zeng, Jun; Peng, Xiaojun; Lu, Xin; Xu, Guowang

    2016-02-05

    The interaction between carbon (C) and nitrogen (N) metabolism can reflect plant growth status and environmental factors. Little is known regarding the connections between C-N metabolism and growing regions under field conditions. To comprehensively investigate the relationship in mature tobacco leaves, we established metabolomics approaches based on gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis-time-of-flight-mass spectrometry (CE-TOF-MS). Approximately 240 polar metabolites were determined. Multivariate statistical analysis revealed that the growing region greatly influenced the metabolic profiles of tobacco leaves. A metabolic correlation network and related pathway maps were used to reveal the global overview of the alteration of C-N metabolism across three typical regions. In Yunnan, sugars and tricarboxylic acid (TCA) cycle intermediates were closely correlated with amino acid pools. Henan tobacco leaves showed positive correlation between the pentose phosphate pathway (PPP) intermediates and C-rich secondary metabolism. In Guizhou, the proline and asparagine had significant links with TCA cycle intermediates and urea cycle, and antioxidant accumulation was observed in response to drought. These results demonstrate that combined analytical approaches have great potential to detect polar metabolites and provide information on C-N metabolism related to planting regional characteristics.

  16. Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in Tac1(-/-) mouse spinal cords reveals significant lower concentration of opioid peptides.

    PubMed

    Saidi, Mouna; Beaudry, Francis

    2015-08-01

    Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1(-/-) spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC-MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1(-/-) mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1(-/-) spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p<0.0001). No significant concentration differences were observed in mouse Tac1(-/-) spinal cords for Met-Enk and CGRP. The analysis of Tac1(-/-) mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

  17. Hydrophilic interaction chromatography based solid-phase extraction and MALDI TOF mass spectrometry for revealing the influence of Pseudomonas fluorescens on phospholipids in salmon fillet.

    PubMed

    Shen, Qing; Yang, Qi; Cheung, Hon-Yeung

    2015-02-01

    Salmon is a popular food but it is easily susceptible to spoilage by contamination with microorganisms. In this study, a method using hydrophilic interaction chromatography (HILIC)-based solid-phase extraction (SPE) and matrix-assisted laser desorption and ionization time-of-flight/time-of-flight mass spectrometry was developed and applied to reveal the effect of Pseudomonas fluorescens on salmon fillet during the shelf-life period by measuring the changes in the levels of phosphatidylcholine and phosphatidylethanolamine. Fresh samples were inoculated with P. fluorescens (10(6) cfu g(-1)) for 30 s, and lipids were extracted at 0, 24, 48, and 72 h. A homemade SPE cartridge packed with HILIC sorbent (silica derivatized with 1,2-dihydroxypropane) was used for matrix cleanup prior to analysis by mass spectrometry. In total, 30 phospholipids and 16 lysophospholipids were detected and elucidated. The results revealed that the content of phospholipids decreased significantly, whereas that of lysophospholipids increased initially, followed by a gradual reduction as the cold storage time increased. The contamination by P. fluorescens negatively affected the quality of fresh salmon without obvious physical changes, but it posed a potential threat to human health. This study suggests that the well-established method could be used for detecting phospholipids in salmon fillet and perhaps other foods as well.

  18. Insulin and epidermal growth factor-urogastrone: Affinity crosslinking to specific binding sites in rat liver membranes

    PubMed Central

    Sahyoun, N.; Hock, R. A.; Hollenberg, M. D.

    1978-01-01

    Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with 125I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with 125I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization. PMID:205865

  19. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  20. A Method Revealing Bacterial Cell-wall Architecture by Time-dependent Isotope Labeling and Quantitative Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Patti, Gary J.; Chen, Jiawei; Gross, Michael L.

    2009-01-01

    The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of post-insertionally modified cell-wall precursors, we find that tripeptides and species without Asx bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens. PMID:19281243

  1. 13C-Tracer and Gas Chromatography-Mass Spectrometry Analyses Reveal Metabolic Flux Distribution in the Oleaginous Microalga Chlorella protothecoides1[C][W][OA

    PubMed Central

    Xiong, Wei; Liu, Lixia; Wu, Chao; Yang, Chen; Wu, Qingyu

    2010-01-01

    The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-13C]/[1-13C]glucose were performed with heterotrophic growth of C. protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. Gas chromatography-mass spectrometry analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle with inactive glyoxylate shunt. Evidence of phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme activity was also obtained. It was demonstrated that the relative activity of the pentose phosphate pathway to glycolysis under nitrogen-limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C. protothecoides remained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on the metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bioproducts. PMID:20720172

  2. Half-width plots, a simple tool to predict peak shape, reveal column kinetics and characterise chromatographic columns in liquid chromatography: state of the art and new results.

    PubMed

    Baeza-Baeza, J J; Ruiz-Ángel, M J; García-Álvarez-Coque, M C; Carda-Broch, S

    2013-11-01

    Peak profiles in chromatography are characterised by their height, position, width and asymmetry; the two latter depend on the values of the left and right peak half-widths. Simple correlations have been found between the peak half-widths and the retention times. The representation of such correlations has been called half-width plots. For isocratic elution, the plots are parabolic, although often, the parabolas can be approximated to straight-lines. The plots can be obtained with the half-widths/retention time data for a set of solutes experiencing the same kinetics, eluted with a mobile phase at fixed or varying composition. When the analysed solutes experience different resistance to mass transfer, the plots will be solute dependent, and should be obtained with the data for each solute eluted with mobile phases at varying composition. The half-width plots approach is a simple tool that facilitates the prediction of peak shape (width and asymmetry) with optimisation purposes, reveal the interaction kinetics of solutes in different columns, and characterise chromatographic columns. This work shows half-width plots for different situations in isocratic elution, including the use of different flows, the effect of temperature, the modification of the stationary phase surface by an additive, the existence of specific interactions within the column, and the comparison of columns. The adaptation to gradient elution is also described. Previous knowledge on half-width plots is structured and analysed, to which new results are added.

  3. Evidence of land plant affinity for the Devonian fossil Protosalvinia (Foerstia)

    USGS Publications Warehouse

    Romankiw, L.A.; Hatcher, P.G.; Roen, J.B.

    1988-01-01

    The Devonian plant fossil Protosalvinia (Foerstia) has been examined by solid-state 13C nuclear magnetic resonance spectroscopy (NMR) and pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). Results of these studies reveal that the chemical structure of Protosalvinia is remarkably similar to that of coalified wood. A well-defined phenolic carbon peak in the NMR spectra and the appearance of phenol and alkylated phenols in pyrolysis products are clearly indicative of lignin-like compounds. These data represent significant new information on the chemical nature of Protosalvinia and provide the first substantial organic geochemical evidence for land plant affinity. -Authors

  4. A dielectric affinity microbiosensor

    NASA Astrophysics Data System (ADS)

    Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

    2010-01-01

    We present an affinity biosensing approach that exploits changes in dielectric properties of a polymer due to its specific, reversible binding with an analyte. The approach is demonstrated using a microsensor comprising a pair of thin-film capacitive electrodes sandwiching a solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid), a synthetic polymer with specific affinity to glucose. Binding with glucose induces changes in the permittivity of the polymer, which can be measured capacitively for specific glucose detection, as confirmed by experimental results at physiologically relevant concentrations. The dielectric affinity biosensing approach holds the potential for practical applications such as long-term continuous glucose monitoring.

  5. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  6. A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

    SciTech Connect

    Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F

    2005-08-25

    Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend

  7. Affine dynamics with torsion

    NASA Astrophysics Data System (ADS)

    Gültekin, Kemal

    2016-03-01

    In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schrödinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor.

  8. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  9. Crystallographic structure of Ni-Co coating on the affinity adsorption of histidine-tagged protein.

    PubMed

    Chang, Yaw-Jen; Chen, Sheng-Zheng; Ho, Ching-Yuan

    2015-04-01

    The principle of immobilized metal affinity chromatography (IMAC) has been recently implemented for protein microarrays for the study of protein abundance and function. Ni-Co film fabricated by electrodeposition is a novel microarray surface in an alloy type for immobilizing histidine-tagged proteins based on IMAC. In this paper, the effects of crystallographic structures and surface properties of Ni-Co coatings, with and without the annealing process, on the immobilization of histidine-tagged proteins were systematically investigated. The experimental results reveal that the stronger hcp texture, due to a higher Co content, results in better affinity adsorption for histidine-tagged biotin. Nevertheless, the allotropic phase transformation from hcp to fcc, due to the annealing process, leads to the decrease of affinity adsorption. The wettability property and the surface roughness of Ni-Co coating are, however, not important factors. Obviously, the crystallographic structure of Ni-Co coating is the dominant factor for the specific affinity adsorption of histidine-tagged protein.

  10. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  11. Applying Chromatography.

    ERIC Educational Resources Information Center

    Klein, Jessie W.; Patev, Paul

    1998-01-01

    Presents three experiments to introduce students to different kinds of chromatography: (1) paper chromatography; (2) gel filtration chromatography; and (3) reverse-phase liquid chromatography. Written in the form of a laboratory manual, explanations of each of the techniques, materials needed, procedures, and a glossary are included. (PVD)

  12. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  13. The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

    PubMed

    Lambrechts, A; Verschelde, J L; Jonckheere, V; Goethals, M; Vandekerckhove, J; Ampe, C

    1997-02-03

    We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

  14. Affine Sphere Relativity

    NASA Astrophysics Data System (ADS)

    Minguzzi, E.

    2017-03-01

    We investigate spacetimes whose light cones could be anisotropic. We prove the equivalence of the structures: (a) Lorentz-Finsler manifold for which the mean Cartan torsion vanishes, (b) Lorentz-Finsler manifold for which the indicatrix (observer space) at each point is a convex hyperbolic affine sphere centered on the zero section, and (c) pair given by a spacetime volume and a sharp convex cone distribution. The equivalence suggests to describe (affine sphere) spacetimes with this structure, so that no algebraic-metrical concept enters the definition. As a result, this work shows how the metric features of spacetime emerge from elementary concepts such as measure and order. Non-relativistic spacetimes are obtained replacing proper spheres with improper spheres, so the distinction does not call for group theoretical elements. In physical terms, in affine sphere spacetimes the light cone distribution and the spacetime measure determine the motion of massive and massless particles (hence the dispersion relation). Furthermore, it is shown that, more generally, for Lorentz-Finsler theories non-differentiable at the cone, the lightlike geodesics and the transport of the particle momentum over them are well defined, though the curve parametrization could be undefined. Causality theory is also well behaved. Several results for affine sphere spacetimes are presented. Some results in Finsler geometry, for instance in the characterization of Randers spaces, are also included.

  15. Neuere Chromatographie

    NASA Astrophysics Data System (ADS)

    Hostettmann, K.

    1983-04-01

    Besides high-performance liquid chromatography (HPLC) which is now a well-established and currently used technique, several emerging methods for the isolation and separation of natural products are receiving considerable attention. Centrifugal thin-layer chromatography is a very rapid technique, but limited in resolution. Of special interest are the recently developed support-free liquid-liquid chromatography methods such as droplet counter-current chromatography (DCCC) and rotation locular counter-current chromatography (RLCC). This latter method was applied to the separation of the enantiomers of (±)-norephedrine.

  16. Striving for Empathy: Affinities, Alliances and Peer Sexuality Educators

    ERIC Educational Resources Information Center

    Fields, Jessica; Copp, Martha

    2015-01-01

    Peer sexuality educators' accounts of their work reveal two approaches to empathy with their students: affinity and alliance. "Affinity-based empathy" rests on the idea that the more commonalities sexuality educators and students share (or perceive they share), the more they will be able to empathise with one another, while…

  17. Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan.

    PubMed

    Ruiz-Bustos, E; Ochoa, J L; Wadström, T; Ascencio, F

    2001-03-01

    A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylori culture supernates was obtained by sequential ammonium sulphate precipitation and affinity chromatography on heparin-Sepharose. The chromatographic procedure yielded one major fraction that contained proteins with heparan sulphate affinity as revealed by inhibition studies of heparan sulphate binding to H. pylori cells. Preparative iso-electric focusing, SDS-PAGE and blotting experiments, with peroxidase(POD)-labelled heparan sulphate as a probe, indicated the presence of two major extracellular proteins with POD-heparan sulphate affinity. One protein had a molecular mass of 66.2 kDa and a pI of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins of H. pylori, whereas the 66.2-kDa HSBP showed a high homology to an Escherichia coli chaperon protein and equine haemoglobin. A third HSBP was isolated from an outer-membrane protein (OMP) fraction of H. pylori cells with a molecular mass of 47.2 kDa. The amino acid sequence of an internal peptide of the OMP-HSBP did not show homology to the extracellular HSBP of H. pylori, or to another microbial HSBP.

  18. Synthesis and characterisation of magnetised Dacron-heparin composite employed for antithrombin affinity purification.

    PubMed

    Mercês, Aurenice Arruda Dutra das; Silva, Ricardo de Souza; Silva, Karciano José Santos; Maciel, Jackeline da Costa; Oliveira, Givanildo Bezerra; Buitrago, Davian Martinez; de Aguiar, José Albino Oliveira; de Carvalho-Júnior, Luiz Bezerra

    2016-12-01

    Human antithrombin is a blood derivative widely used in the treatment of coagulation dysfunction. Affinity chromatography using heparin (HEP) derivatives is usually used for antithrombin purification. In this study, an affinity procedure based on a magnetic Dacron-HEP composite is proposed. Dacron was firstly converted to Dacron-hydrazide and magnetised by co-precipitation with of Fe(2+)/Fe(3+) (mDAC). HEP was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). EDX and infrared spectra analyses confirmed each synthesis step of mDAC-HEP. This composite exhibited superparamagnetism behaviour. Human plasma was incubated with mDAC-HEP (fresh and stored over a long period) and washed with phosphate buffer containing increasing concentrations of NaCl. Human plasma antithrombin activity was reduced by approximately 20% in the presence of the 1.0M NaCl fraction, and this eluate was able to prolong coagulation time (aPTT) using both preparations. Electrophoresis of the eluates revealed bands corresponding to the expected size of antithrombin (58kDa). The mDAC-HEP particles are reusable. This method presents the following advantages: easy, low-cost synthesis of the composite, magnet-based affinity purification steps, and reusability.

  19. Metabolite profiling of Camellia sinensis by automated sequential, multidimensional gas chromatography/mass spectrometry reveals strong monsoon effects on tea constituents.

    PubMed

    Kowalsick, Amanda; Kfoury, Nicole; Robbat, Albert; Ahmed, Selena; Orians, Colin; Griffin, Timothy; Cash, Sean B; Stepp, John Richard

    2014-11-28

    Seasonal variation in tea (Camellia sinensis (L.) Kuntze; Theaceae) chemistry was investigated using automated sequential, multidimensional gas chromatography/mass spectrometry (GC-GC/MS). Metabolite libraries were produced for teas harvested from the Bulang Mountains in Yunnan, China before and after the onset of the East Asian Monsoon. A total of 201 spring and 196 monsoon metabolites were identified, with 169 common and 59 seasonally unique compounds. An additional 163 metabolites were detected but their identity could not be confirmed. Spectral deconvolution of GC/MS data was used to measure the relative concentrations in the teas. Within each family individual metabolite concentrations increased, decreased and stayed the same. The major constituents in both teas were linalool (28%), geraniol (13%), α-terpineol (10%), hotrienol (4%) and nerol (3%). This work provides the foundation to monitor seasonal variations of tea chemistry.

  20. An enhanced plant lipidomics method based on multiplexed liquid chromatography-mass spectrometry reveals additional insights into cold- and drought-induced membrane remodeling.

    PubMed

    Tarazona, Pablo; Feussner, Kirstin; Feussner, Ivo

    2015-11-01

    Within the lipidome of plants a few bulk molecular species hamper the detection of the rest, which are present at relatively low levels. In addition, low-abundance species are often masked by numerous isobaric interferences, such as those caused by isoelemental species and isotopologues. This scenario not only means that minor species are underrepresented, but also leads to potential misidentifications and limits the structural information gathered by lipidomics approaches. In order to overcome these limitations we have developed a multiplexed liquid chromatography-mass spectrometry lipidomics platform able to achieve an enhanced coverage of plant lipidomes. The platform is based on a single extraction step followed by a series of ultra-performance liquid chromatography separations. Post-column flow is then directed to both a triple quadrupole analyzer for targeted profiling and a time-of-flight analyzer for accurate mass analysis. As a proof of concept, plants were subjected to cold or drought, which are known to trigger widespread remodeling events in plant cell membranes. Analysis of the leaf lipidome yielded 393 molecular species within 23 different lipid classes. This enhanced coverage allowed us to identify lipid molecular species and even classes that are altered upon stress, allowing hypotheses on role of glycosylinositolphosphoceramides (GIPC), steryl glycosides (SG) and acylated steryl glycosides (ASG) in drought stress to be addressed and confirming the findings from numerous previous studies with a single, wide-ranging lipidomics approach. This extended our knowledge on membrane remodeling during the drought response, integrating sphingolipids and sterol lipids into the current glycerolipid-based model.

  1. Analysis of biomolecular interactions using affinity microcolumns: A review

    PubMed Central

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L.; White, Christopher J.; Carter, NaTasha; Hage, David S.

    2014-01-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  2. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  3. Gas Chromatography.

    ERIC Educational Resources Information Center

    Karasek, Francis W.; And Others

    1984-01-01

    This review covers fundamental developments in gas chromatography during 1982 and 1983. Literature is considered under these headings: columns; liguid phases; solid supports; sorption processes and solvents; open tubular column gas chromatography; instrumentation; high-resolution columns and applications; other techniques; qualitative and…

  4. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

    PubMed

    Wang, Dongdong; Hincapie, Marina; Rejtar, Tomas; Karger, Barry L

    2011-03-15

    Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace

  5. Studies on human eRF3-PABP interaction reveal the influence of eRF3a N-terminal glycin repeat on eRF3-PABP binding affinity and the lower affinity of eRF3a 12-GGC allele involved in cancer susceptibility

    PubMed Central

    Jerbi, Soumaya; Jolles, Béatrice; Bouceba, Tahar; Jean-Jean, Olivier

    2016-01-01

    ABSTRACT The eukaryotic release factor 3 (eRF3) has been involved in the control of mRNA degradation through its association with the cytoplasmic Poly(A) Binding Protein, PABP. In mammals, eRF3 N-terminal domain contains two overlapping PAM2 motifs which specifically recognize the MLLE domain of PABP. In humans, eRF3a/GSPT1 gene contains a stable GGC repeat encoding a repeat of glycine residues in eRF3a N-terminus. There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines. Several studies have reported that the presence of eRF3a 12-GGC allele is correlated with an increased risk of cancer development. Using surface plasmon resonance, we have studied the interaction of the various allelic forms of eRF3a with PABP alone or poly(A)-bound PABP. We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP. Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation. PMID:26818177

  6. The structure of Erb1-Ytm1 complex reveals the functional importance of a high-affinity binding between two β-propellers during the assembly of large ribosomal subunits in eukaryotes

    PubMed Central

    Wegrecki, Marcin; Rodríguez-Galán, Olga; de la Cruz, Jesús; Bravo, Jeronimo

    2015-01-01

    Ribosome biogenesis is one of the most essential pathways in eukaryotes although it is still not fully characterized. Given the importance of this process in proliferating cells, it is obvious that understanding the macromolecular details of the interactions that take place between the assembly factors, ribosomal proteins and nascent pre-rRNAs is essentially required for the development of new non-genotoxic treatments for cancer. Herein, we have studied the association between the WD40-repeat domains of Erb1 and Ytm1 proteins. These are essential factors for the biogenesis of 60S ribosomal subunits in eukaryotes that form a heterotrimeric complex together with the also essential Nop7 protein. We provide the crystal structure of a dimer formed by the C-terminal part of Erb1 and Ytm1 from Chaetomium thermophilum at 2.1 Å resolution. Using a multidisciplinary approach we show that the β-propeller domains of these proteins interact in a novel manner that leads to a high-affinity binding. We prove that a point mutation within the interface of the complex impairs the interaction between the two proteins and negatively affects growth and ribosome production in yeast. Our study suggests insights into the association of the Erb1-Ytm1 dimer with pre-ribosomal particles. PMID:26476442

  7. The structure of Erb1-Ytm1 complex reveals the functional importance of a high-affinity binding between two β-propellers during the assembly of large ribosomal subunits in eukaryotes.

    PubMed

    Wegrecki, Marcin; Rodríguez-Galán, Olga; de la Cruz, Jesús; Bravo, Jeronimo

    2015-12-15

    Ribosome biogenesis is one of the most essential pathways in eukaryotes although it is still not fully characterized. Given the importance of this process in proliferating cells, it is obvious that understanding the macromolecular details of the interactions that take place between the assembly factors, ribosomal proteins and nascent pre-rRNAs is essentially required for the development of new non-genotoxic treatments for cancer. Herein, we have studied the association between the WD40-repeat domains of Erb1 and Ytm1 proteins. These are essential factors for the biogenesis of 60S ribosomal subunits in eukaryotes that form a heterotrimeric complex together with the also essential Nop7 protein. We provide the crystal structure of a dimer formed by the C-terminal part of Erb1 and Ytm1 from Chaetomium thermophilum at 2.1 Å resolution. Using a multidisciplinary approach we show that the β-propeller domains of these proteins interact in a novel manner that leads to a high-affinity binding. We prove that a point mutation within the interface of the complex impairs the interaction between the two proteins and negatively affects growth and ribosome production in yeast. Our study suggests insights into the association of the Erb1-Ytm1 dimer with pre-ribosomal particles.

  8. Generation of metastatic melanoma specific antibodies by affinity purification

    PubMed Central

    Schütz, Birgit; Koppensteiner, Anita; Schörghofer, David; Kinslechner, Katharina; Timelthaler, Gerald; Eferl, Robert; Hengstschläger, Markus; Missbichler, Albert; Hundsberger, Harald; Mikula, Mario

    2016-01-01

    Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis. PMID:27853253

  9. Kernel Affine Projection Algorithms

    NASA Astrophysics Data System (ADS)

    Liu, Weifeng; Príncipe, José C.

    2008-12-01

    The combination of the famed kernel trick and affine projection algorithms (APAs) yields powerful nonlinear extensions, named collectively here, KAPA. This paper is a follow-up study of the recently introduced kernel least-mean-square algorithm (KLMS). KAPA inherits the simplicity and online nature of KLMS while reducing its gradient noise, boosting performance. More interestingly, it provides a unifying model for several neural network techniques, including kernel least-mean-square algorithms, kernel adaline, sliding-window kernel recursive-least squares (KRLS), and regularization networks. Therefore, many insights can be gained into the basic relations among them and the tradeoff between computation complexity and performance. Several simulations illustrate its wide applicability.

  10. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  11. Functionalized multi-walled carbon nanotubes as affinity ligands

    NASA Astrophysics Data System (ADS)

    Yu, L.; Li, C. M.; Zhou, Q.; Gan, Y.; Bao, Q. L.

    2007-03-01

    Functionalization of carbon nanotubes is very challenging for their applications. The paper here describes a new method to functionalize multi-walled carbon nanotubes (MWCNTs) as specific affinity adsorbents. MWCNTs were acid purified and pretreated with (3-aminopropyl)-triethoxysilane (APTES) in order to introduce abundant amino groups on the surface of MWCNTs. After the conversion of amino groups to carboxyl groups by succinic acid anhydride, MWCNTs were attached to protein A or aminodextran using 1-ethyl-3,3' (dimethylamion)-propylcarbodiimide as a biofunctional crosslinker. The incorporation of aminodextran as a spacer arm noticeably increased the binding capacity of the APTES-modified MWCNTs for protein A. The application of affinity MWCNTs for purification of immunoglobulin G was then evaluated. The affinity of MWCNTs with AMD spacer exhibited a high adsorption capacity of ~361 µg IgG/mg MWCNT (wet basis). About 75% of bound IgG was eluted from affinity MWCNTs (ANT-I and ANT-II) and ELISA confirmed that the biological activity of IgG was well preserved during the course of affinity separation. The functionalized MWCNTs could be potentially used in affinity chromatography.

  12. Gas Chromatography/Mass Spectrometry-Based Metabolomic Profiling Reveals Alterations in Mouse Plasma and Liver in Response to Fava Beans.

    PubMed

    Xiao, Man; Du, Guankui; Zhong, Guobing; Yan, Dongjing; Zeng, Huazong; Cai, Wangwei

    2016-01-01

    Favism is a life-threatening hemolytic anemia resulting from the intake of fava beans by susceptible individuals with low erythrocytic glucose 6-phosphate dehydrogenase (G6PD) activity. However, little is known about the metabolomic changes in plasma and liver after the intake of fava beans in G6PD normal and deficient states. In this study, gas chromatography/mass spectrometry was used to analyze the plasma and liver metabolic alterations underlying the effects of fava beans in C3H- and G6PD-deficient (G6PDx) mice, and to find potential biomarkers and metabolic changes associated with favism. Our results showed that fava beans induced oxidative stress in both C3H and G6PDx mice. Significantly, metabolomic differences were observed in plasma and liver between the control and fava bean treated groups of both C3H and G6PDx mice. The levels of 7 and 21 metabolites in plasma showed significant differences between C3H-control (C3H-C)- and C3H fava beans-treated (C3H-FB) mice, and G6PDx-control (G6PDx-C)- and G6PDx fava beans-treated (G6PDx-FB) mice, respectively. Similarly, the levels of 7 and 25 metabolites in the liver showed significant differences between C3H and C3H-FB, and G6PDx and G6PDx-FB, respectively. The levels of oleic acid, linoleic acid, and creatinine were significantly increased in the plasma of both C3H-FB and G6PDx-FB mice. In the liver, more metabolic alterations were observed in G6PDx-FB mice than in C3H-FB mice, and were involved in a sugar, fatty acids, amino acids, cholesterol biosynthesis, the urea cycle, and the nucleotide metabolic pathway. These findings suggest that oleic acid, linoleic acid, and creatinine may be potential biomarkers of the response to fava beans in C3H and G6PDx mice and therefore that oleic acid and linoleic acid may be involved in oxidative stress induced by fava beans. This study demonstrates that G6PD activity in mice can affect their metabolic pathways in response to fava beans.

  13. Gas Chromatography/Mass Spectrometry-Based Metabolomic Profiling Reveals Alterations in Mouse Plasma and Liver in Response to Fava Beans

    PubMed Central

    Zhong, Guobing; Yan, Dongjing; Zeng, Huazong; Cai, Wangwei

    2016-01-01

    Favism is a life-threatening hemolytic anemia resulting from the intake of fava beans by susceptible individuals with low erythrocytic glucose 6-phosphate dehydrogenase (G6PD) activity. However, little is known about the metabolomic changes in plasma and liver after the intake of fava beans in G6PD normal and deficient states. In this study, gas chromatography/mass spectrometry was used to analyze the plasma and liver metabolic alterations underlying the effects of fava beans in C3H- and G6PD-deficient (G6PDx) mice, and to find potential biomarkers and metabolic changes associated with favism. Our results showed that fava beans induced oxidative stress in both C3H and G6PDx mice. Significantly, metabolomic differences were observed in plasma and liver between the control and fava bean treated groups of both C3H and G6PDx mice. The levels of 7 and 21 metabolites in plasma showed significant differences between C3H-control (C3H-C)- and C3H fava beans-treated (C3H-FB) mice, and G6PDx-control (G6PDx-C)- and G6PDx fava beans-treated (G6PDx-FB) mice, respectively. Similarly, the levels of 7 and 25 metabolites in the liver showed significant differences between C3H and C3H-FB, and G6PDx and G6PDx-FB, respectively. The levels of oleic acid, linoleic acid, and creatinine were significantly increased in the plasma of both C3H-FB and G6PDx-FB mice. In the liver, more metabolic alterations were observed in G6PDx-FB mice than in C3H-FB mice, and were involved in a sugar, fatty acids, amino acids, cholesterol biosynthesis, the urea cycle, and the nucleotide metabolic pathway. These findings suggest that oleic acid, linoleic acid, and creatinine may be potential biomarkers of the response to fava beans in C3H and G6PDx mice and therefore that oleic acid and linoleic acid may be involved in oxidative stress induced by fava beans. This study demonstrates that G6PD activity in mice can affect their metabolic pathways in response to fava beans. PMID:26981882

  14. Gas Chromatography.

    ERIC Educational Resources Information Center

    Cram, Stuart P.; And Others

    1980-01-01

    Selects fundamental developments in theory, methodology, and instrumentation in gas chromatography (GC). A special section reviews GC in the People's Republic of China. Over 1,000 references are cited. (CS)

  15. O(2)-dependent K(+) fluxes in trout red blood cells: the nature of O(2) sensing revealed by the O(2) affinity, cooperativity and pH dependence of transport.

    PubMed

    Berenbrink, M; Völkel, S; Heisler, N; Nikinmaa, M

    2000-07-01

    The effects of pH and O(2) tension on the isotonic ouabain-resistant K(+) (Rb+) flux pathway and on haemoglobin O2 binding were studied in trout red blood cells (RBCs) in order to test for a direct effect of haemoglobin O(2) saturation on K(+) transport across the RBC membrane. At pH values corresponding to in vivo control arterial plasma pH and higher, elevation of the O(2) partial pressure (PO(2)) from 7.8 to 157 mmHg increased unidirectional K(+) influx across the RBC membrane several-fold. At lower extracellular pH values, stimulation of K(+) influx by O(2) was depressed, exhibiting an apparent pK(a) (pK'(a)) for the process of 8.0. Under similar conditions the pK'(a) for acid-induced deoxygenation of haemoglobin (Hb) was 7.3. When trout RBCs were exposed to PO(2) values between 0 and 747 mmHg, O(2) equilibrium curves typical of Hb O(2) saturation were also obtained for K(+) influx and efflux. However, at pH 7.9, the PO(2) for half-maximal K(+) efflux and K(+) influx (P50) was about 8- to 12-fold higher than the P(50) for Hb-O(2) binding. While K(+) influx and efflux stimulation by O(2) was essentially non-cooperative, Hb-O(2) equilibrium curves were distinctly sigmoidal (Hill parameters close to 1 and 3, respectively). O(2)-stimulated K(+) influx and efflux were strongly pH dependent. When the definition of the Bohr factor for respiratory pigments (Phi = delta logP50 x delta pH(-1)) was extended to the effect of pH on O(2)-dependent K(+) influx and efflux, extracellular Bohr factors (Phi(o) of -2.00 and -2.06 were obtained, values much higher than that for Hb (Phi(o) = -0.49). The results of this study are consistent with an O(2) sensing mechanism differing markedly in affinity and cooperativity of O(2) binding, as well as in pH sensitivity, from bulk Hb.

  16. Validation of affinity reagents using antigen microarrays.

    PubMed

    Sjöberg, Ronald; Sundberg, Mårten; Gundberg, Anna; Sivertsson, Asa; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter

    2012-06-15

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  17. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  18. Electrospun polyethersulfone affinity membrane: membrane preparation and performance evaluation.

    PubMed

    Ma, Zuwei; Lan, Zhengwei; Matsuura, Takeshi; Ramakrishna, Seeram

    2009-11-01

    Non-woven polyethersulfone (PES) membranes were prepared by electrospinning. After heat treatment and surface activation, the membranes were covalently functionalized with ligands to be used as affinity membranes. The membranes were characterized in terms of fiber diameter, porosity, specific area, pore size, ligand density and binding capacities. To evaluate the binding efficiency of the membrane, dynamic adsorption of bovine serum albumin (BSA) on the Cibacron blue F3GA (CB) functionalized PES membrane was studied. Experimental breakthrough curves were fitted with the theoretical curves based on the plate model to estimate plate height (H(p)) of the affinity membrane. The high value of H(p) (1.6-8 cm) of the affinity membrane implied a poor dynamic binding efficiency, which can be explained by the intrinsic microstructures of the material. Although the electrospun membrane might not be an ideal candidate for the preparative affinity membrane chromatography for large-scale production, it still can be used for fast small-scale protein purification in which a highly efficient binding is not required. Spin columns packed with protein A/G immobilized PES membranes were demonstrated to be capable of binding IgG specifically. SDS-PAGE results demonstrated that the PES affinity membrane had high specific binding selectivity for IgG molecules and low non-specific protein adsorption. Compared with other reported affinity membranes, the PES affinity membrane had a comparable IgG binding capacity of 4.5 mg/ml, and had a lower flow through pressure drop due to its larger pore size. In conclusion, the novel PES affinity membrane is an ideal spin column packing material for fast protein purification.

  19. Affinity-based target deconvolution of safranal

    PubMed Central

    2013-01-01

    Background and the purpose of the study Affinity-based target deconvolution is an emerging method for the identification of interactions between drugs/drug candidates and cellular proteins, and helps to predict potential activities and side effects of a given compound. In the present study, we hypothesized that a part of safranal pharmacological effects, one of the major constituent of Crocus sativus L., relies on its physical interaction with target proteins. Methods Affinity chromatography solid support was prepared by covalent attachment of safranal to agarose beads. After passing tissue lysate through the column, safranal-bound proteins were isolated and separated on SDS-PAGE or two-dimensional gel electrophoresis. Proteins were identified using MALDI-TOF/TOF mass spectrometry and Mascot software. Results and major conclusion Data showed that safranal physically binds to beta actin, cytochrome b-c1 complex sub-unit 1, trifunctional enzyme sub-unit beta and ATP synthase sub-unit alpha and beta. These interactions may explain part of safranal’s pharmacological effects. However, phenotypic and/or biological relevance of these interactions remains to be elucidated by future pharmacological studies. PMID:23514587

  20. Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

    PubMed

    Parte, Priyanka P; Rao, Parimala; Redij, Shweta; Lobo, Vivian; D'Souza, Serena J; Gajbhiye, Rahul; Kulkarni, Vijay

    2012-10-22

    Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.

  1. Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

    PubMed Central

    de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra

    2016-01-01

    Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

  2. Green chromatography.

    PubMed

    Płotka, Justyna; Tobiszewski, Marek; Sulej, Anna Maria; Kupska, Magdalena; Górecki, Tadeusz; Namieśnik, Jacek

    2013-09-13

    Analysis of organic compounds in samples characterized by different composition of the matrix is very important in many areas. A vast majority of organic compound determinations are performed using gas or liquid chromatographic methods. It is thus very important that these methods have negligible environmental impact. Chromatographic techniques have the potential to be greener at all steps of the analysis, from sample collection and preparation to separation and final determination. The paper summarizes the approaches used to accomplish the goals of green chromatography. While complete elimination of sample preparation would be an ideal approach, it is not always practical. Solventless extraction techniques offer a very good alternative. Where solvents must be used, the focus should be on the minimization of their consumption. The approaches used to make chromatographic separations greener differ depending on the type of chromatography. In gas chromatography it is advisable to move away from using helium as the carrier gas because it is a non-renewable resource. GC separations using low thermal mass technology can be greener because of energy savings offered by this technology. In liquid chromatography the focus should be on the reduction of solvent consumption and replacement of toxic and environmentally hazardous solvents with more benign alternatives. Multidimensional separation techniques have the potential to make the analysis greener in both GC and LC. The environmental impact of the method is often determined by the location of the instrument with respect to the sample collection point.

  3. Ion Chromatography.

    ERIC Educational Resources Information Center

    Mulik, James D.; Sawicki, Eugene

    1979-01-01

    Accurate for the analysis of ions in solution, this form of analysis enables the analyst to directly assay many compounds that previously were difficult or impossible to analyze. The method is a combination of the methodologies of ion exchange, liquid chromatography, and conductimetric determination with eluant suppression. (Author/RE)

  4. Contractions of affine spherical varieties

    SciTech Connect

    Arzhantsev, I V

    1999-08-31

    The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL{sub 2}-varieties are considered.

  5. Gas chromatography.

    PubMed

    Eiceman, G A; Hill, H H; Gardea-Torresdey, J

    1998-06-15

    This review of the fundamental developments in gas chromatography (GC) includes articles published from 1996 and 1997 and an occasional citation prior to 1996. The literature was reviewed principally using CA Selects for Gas Chromatography from Chemical Abstracts Service, and some significant articles from late 1997 may be missing from the review. In addition, the online SciSearch Database (Institute for Scientific Information) capability was used to abstract review articles or books. As with the prior recent reviews, emphasis has been given to the identification and discussion of selected developments, rather than a presentation of a comprehensive literature search, now available widely through computer-based resources. During the last two years, several themes emerged from a review of the literature. Multidimensional gas chromatography has undergone transformation encompassing a broad range of activity, including attempts to establish methods using chromatographic principles rather than a totally empirical approach. Another trend noted was a comparatively large effort in chromatographic theory through modeling efforts; these presumably became resurgent with inexpensive and powerful computing tools. Finally, an impressive level of activity was noted through the themes highlighted in this review, and this was particularly true with detectors and field instruments.

  6. /Chromatography+RECOVERY=superresolution chromatography

    NASA Astrophysics Data System (ADS)

    Kosarev, E. L.; Muranov, K. O.

    2003-04-01

    A method for improving the resolution of the chromatographic analysis based on deriving the point-spread function of a chromatographic column, i.e., a chromatogram of an individual compound, is described. The system of two data sets, namely, a chromatogram of a substance analyzed and a point-spread function of a chromatographic column in combination with the noise statistics, makes it possible to use the RECOVERY signal-reconstruction software package described in paper by Gelfgat et al. (Comp. Phys. Commun. 74 (1993) 335). The proposed method has been tested by chromatography of bovine serum albumin using gel filtration. The resultant resolution exceeds that reached using high-performance liquid chromatography (with the cost of the instruments being lower by a factor of 15-20).

  7. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

  8. Are axial and radial flow chromatography different?

    PubMed

    Besselink, Tamara; van der Padt, Albert; Janssen, Anja E M; Boom, Remko M

    2013-01-04

    Radial flow chromatography can be a solution for scaling up a packed bed chromatographic process to larger processing volumes. In this study we compared axial and radial flow affinity chromatography both experimentally and theoretically. We used an axial flow column and a miniaturized radial flow column with a ratio of 1.8 between outer and inner surface area, both with a bed height of 5 cm. The columns were packed with affinity resin to adsorb BSA. The average velocity in the columns was set equal. No difference in performance between the two columns could be observed. To gain more insight into the design of a radial flow column, the velocity profile and resin distribution in the radial flow column were calculated. Using mathematical models we found that the breakthrough performance of radial flow chromatography is very similar to axial flow when the ratio between outer and inner radius of the radial flow column is around 2. When this ratio is increased, differences become more apparent, but remain small. However, the ratio does have a significant influence on the velocity profile inside the resin bed, which directly influences the pressure drop and potentially resin compression, especially at higher values for this ratio. The choice between axial and radial flow will be based on cost price, footprint and packing characteristics. For small-scale processes, axial flow chromatography is probably the best choice, for resin volumes of at least several tens of litres, radial flow chromatography may be preferable.

  9. Chemical binding affinity estimation using MSB

    NASA Astrophysics Data System (ADS)

    Weaver, John B.; Rauwerdink, Adam M.

    2011-03-01

    Binding affinity can be estimated in several ways in the laboratory but there is no viable way to estimate binding affinity in vivo without assumptions on the number of binding sites. Magnetic spectroscopy of nanoparticle Brownian motion, MSB, measures the rotational Brownian motion. The MSB signal is affected by nanoparticle binding affinity so it provides a mechanism to measure the chemical binding affinity. We present a possible mechanism to quantify the binding affinity and test that mechanism using viscous solutions.

  10. Isolation of murine sialoglycoprotein using consecutive chromatography.

    PubMed

    Wilson, D J; Planas, J M

    1991-01-01

    Affinity columns and high performance liquid chromatography were employed consecutively to obtain 89, 65, 46 and 29 kilodalton sialoglycoproteins from mouse erythrocyte ghosts free of the Band 3 protein which traditionally co-purifies with these proteins. The purification scheme involves Concanavalin A, Wheat Germ Agglutinin and/or Limulus lectin Sepharose 4B columns. We have designated these glycophorin-like proteins Sialoglycoproteins 1, 2, 3, and 4, respectively. Sialoglycoprotein 2 can be isolated independently using a Limulus column combination, while Sialoglycoproteins 3 and 4 were isolated separately during high performance liquid chromatography, demonstrating heterogeneity in binding properties between these sialoglycoproteins.

  11. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  12. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  13. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  14. ELECTRON AFFINITIES OF INORGANIC RADICALS.

    DTIC Science & Technology

    energy in the latter compound is 110 kcals/mole, distinctly higher than in ammonia. Cyanogen (CN)2 and hydrocyanic acid (HCN) yield values for the...ions very readily, and the electron affinity is 49 kcals/mole. A comparison with the results from thiocyanic acid (HNCS) indicates that the H-N bond

  15. Gas Chromatography

    NASA Astrophysics Data System (ADS)

    Qian, Michael C.

    Gas chromatography (GC) has many applications in the analysis of food products. GC has been used for the determination of fatty acids, triglycerides, cholesterol, gases, water, alcohols, pesticides, flavor compounds, and many more. While GC has been used for other food components such as sugars, oligosaccharides, amino acids, peptides, and vitamins, these substances are more suited to analysis by high performance liquid chromatography. GC is ideally suited to the analysis of volatile substances that are thermally stable. Substances such as pesticides and flavor compounds that meet these criteria can be isolated from a food and directly injected into the GC. For compounds that are thermally unstable, too low in volatility, or yield poor chromatographic separation due to polarity, a derivatization step must be done before GC analysis. The two parts of the experiment described here include the analysis of alcohols that requires no derivatization step, and the analysis of fatty acids which requires derivatization. The experiments specify the use of capillary columns, but the first experiment includes conditions for a packed column.

  16. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems.

  17. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    SciTech Connect

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. )

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  18. A new alpha-globin variant with increased oxygen affinity in a Swiss family: Hb Frauenfeld [alpha 138(H21)Ser-->Phe, TCC>TTC (alpha 2)].

    PubMed

    Hochuli, Michel; Zurbriggen, Karin; Schmid, Marlis; Speer, Oliver; Rochat, Philippe; Frauchiger, Beat; Kleinert, Peter; Schmugge, Markus; Troxler, Heinz

    2009-01-01

    A new alpha-globin mutation [alpha 138(H21)Ser-->Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an alpha-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the alpha2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser-->Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.

  19. Centrifugal precipitation chromatography.

    PubMed

    Ito, Yoichiro; Qi, Lin

    2010-01-15

    Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.

  20. Generation of an affinity column for antibody purification by intein-mediated protein ligation.

    PubMed

    Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2003-11-01

    Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

  1. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  2. Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.

    PubMed

    Panwar, Pankaj; Deniaud, Aurélien; Pebay-Peyroula, Eva

    2012-10-01

    Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 Å resolution of Strep-Tactin in complex with desthiobiotin.

  3. Instrumentation: Ion Chromatography.

    ERIC Educational Resources Information Center

    Fritz, James S.

    1987-01-01

    Discusses the importance of ion chromatography in separating and measuring anions. The principles of ion exchange are presented, along with some applications of ion chromatography in industry. Ion chromatography systems are described, as well as ion pair and ion exclusion chromatography, column packings, detectors, and programming. (TW)

  4. High-throughput fragment screening by affinity LC-MS.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in <4 h (corresponding to >3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  5. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.

  6. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling.

    PubMed

    Hackel, Benjamin J; Kapila, Atul; Wittrup, K Dane

    2008-09-19

    The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino acid composition and loop length. The library was screened by fluorescence-activated cell sorting to isolate binders to lysozyme. An affinity maturation scheme was developed to rapidly and broadly diversify populations of clones by random mutagenesis as well as homologous recombination-driven shuffling of mutagenized loops. The novel library and affinity maturation scheme combined to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A secondary affinity maturation identified a stable 1.1 pM binder, the highest affinity yet reported for an Fn3 domain. In addition to extension of the affinity limit for this scaffold, the results demonstrate the ability to achieve high-affinity binding while preserving stability and the monomeric state. This library design and affinity maturation scheme is highly efficient, utilizing an initial diversity of 2x10(7) clones and screening only 1x10(8) mutants (totaled over all affinity maturation libraries). Analysis of intermediate populations revealed that loop length diversity, loop shuffling, and recursive mutagenesis of diverse populations are all critical components.

  7. A luminescent affinity tag for proteins based on the terbium(III)-binding peptide.

    PubMed

    Sueda, Shinji; Tanaka, Shogo; Inoue, Sayomi; Komatsu, Hideyuki

    2012-03-01

    Genetically encoded tags attached to proteins of interest are widely exploited for proteome analysis. Here, we present Tb(3+)-binding peptides (TBPs) which can be used for both luminescent measurements and affinity purification of proteins. TBPs consist of acidic amino acid residues and tryptophan residues which serve as Tb(3+)-binding sites and sensitizers for Tb(3+) luminescence, respectively. The Tb(3+) complexes of TBPs fused to a target protein exhibited luminescence characteristic of Tb(3+) by excitation of the tryptophan residue, and fusion proteins fused to one of the TPBs were successfully isolated from Escherichia coli cell lysate by affinity chromatography with a Tb(3+)-immobilized solid support.

  8. Highly efficient and low-cost purification of lysozyme: a novel tris(hydroxymethyl)aminomethane immobilized affinity column.

    PubMed

    Quan, Li; Cao, Qing; Li, Zhiyu; Li, Na; Li, Kean; Liu, Feng

    2009-03-01

    A highly efficient and low-cost affinity chromatography strategy for lysozyme (LZM) purification is reported. Using tris(hydroxymethyl)aminomethane (Tris) as ligand and macroporous silica spheres as matrix, a novel affinity column was prepared. The high specificity, stability and repeatability of this Tris immobilized affinity column were proved by LZM separations from protein mixture solutions for 20 circles and 6 months test. LZM purified from chicken egg white on the Tris affinity column had even higher purity than the commercial standard and well-maintained activity of 8287 U/mg (activity of commercial LZM was 8171 U/mg). The efficient affinity process avoiding expensive or fragile ligand would bring advantages to the routine production of LZM from chicken egg white.

  9. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  10. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    PubMed Central

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  11. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  12. Affinity membrane introduction mass spectrometry

    SciTech Connect

    Xu, C.; Patrick, J.S.; Cooks, R.G. )

    1995-02-15

    A new technique, affinity membrane introduction mass spectrometry, is described. In this method, a chemically modified membrane is used to selectively adsorb analytes bearing a particular functional group and concentrate them from solution. Release of the bound analyte results in its transfer across the membrane and allows it to be monitored mass spectrometrically, using, in the present case, a benchtop ion trap instrument. Alkylamine-modified cellulose membranes are used to bind substituted benzaldehydes through imine formation at high pH. Release of the bound aldehyde is achieved by acid hydrolysis of the surface-bound imine. Benzaldehyde is detected with excellent specificity at 10 ppm in a complex mixture using this method. Using the enrichment capability of the membrane, a full mass spectrum of benzaldehyde can be measured at a concentration of 10 ppb. The behavior of a variety of other aldehydes is also discussed to illustrate the capabilities of the method. 21 refs., 5 figs., 2 tabs.

  13. Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin.

    PubMed

    Brophy, Megan Brunjes; Nakashige, Toshiki G; Gaillard, Aleth; Nolan, Elizabeth M

    2013-11-27

    Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96-114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103-105 to 104-106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

  14. Antisymmetric tensor generalizations of affine vector fields

    PubMed Central

    Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-01-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank-p antisymmetric affine tensor fields in n-dimensions is bounded by (n + 1)!/p!(n − p)!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes. PMID:26858463

  15. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, Kenneth Sherman

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  16. Basic Principles of Chromatography

    NASA Astrophysics Data System (ADS)

    Ismail, Baraem; Nielsen, S. Suzanne

    Chromatography has a great impact on all areas of analysis and, therefore, on the progress of science in general. Chromatography differs from other methods of separation in that a wide variety of materials, equipment, and techniques can be used. [Readers are referred to references (1-19) for general and specific information on chromatography.]. This chapter will focus on the principles of chromatography, mainly liquid chromatography (LC). Detailed principles and applications of gas chromatography (GC) will be discussed in Chap. 29. In view of its widespread use and applications, high-performance liquid chromatography (HPLC) will be discussed in a separate chapter (Chap. 28). The general principles of extraction are first described as a basis for understanding chromatography.

  17. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  18. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  19. Dynamic friction of self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Schmittbuhl, Jean; Vilotte, Jean-Pierre; Roux, Stéphane

    1994-02-01

    We investigate the velocity dependence of the friction between two rigid blocks limited by a self-affine surface such as the one generated by a crack. The upper solid is subjected either to gravity or to an external elastic stiffness, and is driven horizontally at constant velocity, V, while the lower solid is fixed. For low velocities, the apparent friction coefficient is constant. For high velocities, the apparent friction is shown to display a velocity weakening. The weakening can be related to the variation of the mean contact time due to the occurrence of jumps during the motions. The cross-over between these two regimes corresponds to a characteristic velocity which depends on the geometry of the surfaces and on the mean normal force. In the case of simple gravity loading, the velocity dependence of the apparent friction at high velocities is proportional to 1/V^2 where V is the imposed tangential velocity. In the case of external elastic stiffness, two velocity weakening regimes can be identified, the first is identical to the gravity case with a 1/V^2 dependence, the second appears at higher velocities and is characterized by a 1/V variation. The characteristic velocity of this second cross-over depends on the roughness and the elastic stiffness. The statistical distribution of ballistic flight distances is analysed, and is shown to reveal in all cases the self-affinity of the contacting surfaces. Nous analysons la dépendence en vitesse du frottement entre deux solides limités par une surface rugueuse auto-affine comme celle d'une surface de fracture. Le solide supérieur est soumis soit à la gravité, soit à une raideur élastique externe, et est entraîné à vitesse horizontale constante V sur le solide inférieur fixe. A faible vitesse, le coefficient de friction apparent, est constant. A forte vitesse, le coefficient de friction apparent devient inversement proportionnel à la vitesse. Cette dépendance peut être reliée à la variation du temps

  20. Chromatography: concepts and contrasts

    SciTech Connect

    Miller, J.M.

    1988-01-01

    As the author states in the Preface, this text attempts to provide a unified approach to chromatography (hence the title) by way of contrasting similarities and differences between gas chromatography (GC), column liquid chromatography (LC), and thin-layer chromatography (TLC). This book is also said to be pitched at an elementary level, suitable for most newcomers to the field (e.g., advanced undergraduates and beginning graduate students in the academic world, as well as bench-level chemists in industry).

  1. A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with differ...

  2. Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation.

    PubMed

    Fields, Conor; Li, Peng; O'Mahony, James J; Lee, Gil U

    2016-01-01

    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future.

  3. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  4. Cation exchange displacement batch chromatography of proteins guided by screening of protein purification parameters.

    PubMed

    Kotasińska, Marta; Richter, Verena; Thiemann, Joachim; Schlüter, Hartmut

    2012-11-01

    Displacement chromatography has been shown to be an effective alternative for protein purification. We investigated in this study sample displacement chromatography, which does not require a displacer molecule. Furthermore, we performed a screening for determination of parameters for an optimal sample displacement chromatography. We screened the affinities of cytochrome C, lysozyme, myoglobin, and ribonuclease A toward a cation exchange material as a function of different pH values and to presence of different concentrations of sodium chloride in the sample application buffer. Sample displacement chromatography in batch chromatography mode for the separation of the protein mixture was studied with a sample application buffer with a pH of 5 and 7. As predicted by the screening experiments, sample displacement chromatography was most effective at pH 7 since this pH guaranteed the largest differences of the affinities of the four proteins toward the stationary phase. In summary, we describe here sample displacement chromatography in the batch chromatography mode for the separation of proteins, which is a simple and fast alternative to conventional displacement chromatography. Systematic screening of chromatographic parameters prior to sample displacement chromatography promises a successful separation of a target protein.

  5. Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation.

    PubMed

    Keck, Simone; Schmaler, Mathias; Ganter, Stefan; Wyss, Lena; Oberle, Susanne; Huseby, Eric S; Zehn, Dietmar; King, Carolyn G

    2014-10-14

    Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (TFH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the TFH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.

  6. Preparation and evaluation of Ricinus communis agglutinin affinity adsorbents using polymeric supports.

    PubMed

    Cartellieri, S; Helmholz, H; Niemeyer, B

    2001-08-01

    A practicable and efficient procedure for preparation of Ricinus communis agglutinin (RCA) affinity adsorbents has been developed. For immobilization of RCA two different polymer-based supports, Toyopearl and TSKgel (TosoHaas), were used. RCA has been successfully immobilized onto these supports with amounts of coupled ligand between 15 and 23 mg/g dry support and corresponding coupling yields of 69-93% (w/w). The prepared affinity adsorbents were characterized concerning their binding capacity for the glycoprotein asialofetuin (ASF) and accessibility of the ligand binding sites. The high accessibility of 80% showed that steric hindrance was negligible at the present ligand density. RCA-Toyopearl was successfully applied in affinity chromatography of glycoproteins indicating its high specificity. A long-term stability test proved no change in capacity for a period of at least 12 months. High-performance affinity chromatography (HPLAC) was carried out using RCA-TSKgel. Experimental results showed that the prepared adsorbents are suitable for selective separation of glycoproteins and oligosaccharides and therefore can be used for investigations of adsorption characteristics of glycoconjugates and for laboratory-scale preparations.

  7. Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

    PubMed Central

    Devey, M E; Major, P J; Bleasdale-Barr, K M; Holland, G P; Dal Canto, M C; Paterson, P Y

    1990-01-01

    Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post-sensitization. Antibodies to myelin basic protein (MBP) were detected in both lines but rising titres of high functional affinity antibodies were only seen in HA mice. These HA and LA lines of mice provide a new approach to the study of EAE and, in particular, the role of antibody and antibody affinity in the chronic relapsing form of the disease. Images Figure 2 PMID:2335373

  8. Bioengineering of bacteria to assemble custom-made polyester affinity resins.

    PubMed

    Hay, Iain D; Du, Jinping; Burr, Natalie; Rehm, Bernd H A

    2015-01-01

    Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.

  9. Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

    PubMed Central

    Hay, Iain D.; Du, Jinping; Burr, Natalie

    2014-01-01

    Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

  10. Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

    PubMed

    Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan

    2014-07-22

    Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV.

  11. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    PubMed

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  12. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  13. Protein A affinity precipitation of human immunoglobulin G.

    PubMed

    Janoschek, Lars; Freiherr von Roman, Matthias; Berensmeier, Sonja

    2014-08-15

    The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit(®) S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1°C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes.

  14. Improved Thermal Modulator for Gas Chromatography

    NASA Technical Reports Server (NTRS)

    Hasselbrink, Ernest Frederick, Jr.; Hunt, Patrick J.; Sacks, Richard D.

    2008-01-01

    An improved thermal modulator has been invented for use in a variant of gas chromatography (GC). The variant in question denoted as two-dimensional gas chromatography (2DGC) or GC-GC involves the use of three series-connected chromatographic columns, in the form of capillary tubes coated interiorly with suitable stationary phases (compounds for which different analytes exhibit different degrees of affinity). The two end columns are relatively long and are used as standard GC columns. The thermal modulator includes the middle column, which is relatively short and is not used as a standard GC column: instead, its temperature is modulated to affect timed adsorption and desorption of analyte gases between the two end columns in accordance with a 2DGC protocol.

  15. Structure of Greyhound hemoglobin: origin of high oxygen affinity.

    PubMed

    Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

    2011-05-01

    This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

  16. Affinity separation of human plasma gelsolin on Affi-Gel Blue.

    PubMed

    Yamamoto, H; Terabayashi, M; Egawa, T; Hayashi, E; Nakamura, H; Kishimoto, S

    1989-05-01

    Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.

  17. Chromatography resin support

    DOEpatents

    Dobos, James G.

    2002-01-01

    An apparatus and method of using an improved chromatography resin support is disclosed. The chromatography support platform is provided by a stainless steel hollow cylinder adapted for being inserted into a chromatography column. An exterior wall of the stainless steel cylinder defines a groove for carrying therein an "O"-ring. The upper surface of the stainless steel column is covered by a fine stainless steel mesh welded to the edges of the stainless steel cylinder. When placed upon a receiving ledge defined within a chromatography column, the "O"-ring provides a fluid tight seal with the inner edge wall of the chromatography cylinder. The stainless steel mesh supports the chromatography matrix and provides a back flushable support which is economical and simple to construct.

  18. An online coupled peritoneal macrophage/cell membrane chromatography and high-performance liquid chromatography/mass spectrometry method to screen for anti-inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei.

    PubMed

    Li, Weifeng; Xing, Wei; Wang, Sicen; Fan, Ting; Huang, Huimin; Niu, Xiaofeng; He, Langchong

    2013-11-01

    Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti-inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor-α in lipopolysaccharide-stimulated mice and peritoneal macrophages in a dose-dependent manner. The results demonstrated that the PM/CMC-online-HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples.

  19. Anacardium occidentale bark lectin: purification, immobilization as an affinity model and influence in the uptake of technetium-99M by rat adipocytes.

    PubMed

    Maciel, Maria Inês Sucupira; de Mendonça Cavalcanti, Maria do Socorro; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; de Almeida Catanho, Maria Teresa Jansem; Coelho, Luana Cassandra Breitenbach Barroso

    2012-10-01

    Lectins, proteins that recognize carbohydrates, have been immobilized on inert supports and used in the screening or purification of glycoproteins. Anacardium occidentale bark infusion has been used as a hypoglycemic agent in Brazil. The toxicity of natural products may be evaluated determining their capability to alter the biodistribution of technetium-99M ((99m)Tc). This work reports the isolation and characterization of a lectin from A. occidentale bark (AnocBL), its evaluation as an affinity support for glycoprotein isolation and lectin effect on the uptake of (99m)Tc by rat adipocytes. AnocBL was isolated from 80 % ammonium sulphate supernatant by affinity chromatography on fetuin-agarose. SDS-PAGE showed a single protein band of 47 kDa. The monossacharide L-arabinose and the glycoproteins fetuin, asialofetuin, ovomucoid, casein, thyroglobulin, peroxidase, fetal bovine serum and IgG inhibited the activity. The lectin activity was stable until 70 °C and at a pH range of 3.0-7.5. AnocBL-Sepharose column bound fetuin indicating that the lectin matrix may be used to obtain glycoconjugates of biotechnological interest. In vitro assay revealed that glucose and insulin increase (99m)Tc uptake by rat adipocytes. AnocBL decreases (99m)Tc uptake, and this effect was not detected in the presence of glucose. Fetuin inhibited AnocBL effect in all insulin concentrations.

  20. Dual-Metal Centered Zirconium-Organic Framework: A Metal-Affinity Probe for Highly Specific Interaction with Phosphopeptides.

    PubMed

    Peng, Jiaxi; Zhang, Hongyan; Li, Xin; Liu, Shengju; Zhao, Xingyun; Wu, Jing; Kang, Xiaohui; Qin, Hongqiang; Pan, Zaifa; Wu, Ren'an

    2016-12-28

    The highly specific affinity between probes and phosphopeptides is the fundamental interaction for selective identification of phosphoproteomes that uncover the mechanisms of signal transduction, cell cycle, enzymatic regulation, and gene expression in biological systems. In this study, a metal-affinity probe possessing both interactions of metal oxide affinity chromatography (MOAC) and immobilized metal ion affinity chromatography (IMAC) was facilely prepared by immobilizing zirconium(IV) on a zirconium-organic framework of UiO-66-NH2, which holds dual-metal centers of not only the inherent Zr-O cluster but also the immobilized Zr(IV) center. This dual-metal centered zirconium-organic framework (DZMOF) demonstrates as a highly specific metal-affinity probe toward the extraction of phosphopeptides due to the metal-affinity interactions of MOAC and IMAC toward either mono-phosphorylated or multi-phosphorylated peptides. The binding energies of zirconium 3d5/2 and 3d3/2 in this DZMOF are 183.07 and 185.47 eV, respectively, which are higher than those of the intact UiO-66-NH2 (182.84 and 185.17 eV, respectively), confirming the higher metal-affinity interaction between the DZMOF and phosphopeptides. This high metal-affinity probe presents an unprecedented strong performance in anti-nonspecific interference during the capturing of phosphopeptides of β-casein with the molar ratio of β-casein vs bovine serum albumin up to ca. 1:5000. The enrichment of phosphopeptides from a human saliva sample by DZMOF further confirms the great potential of DZMOF in the extraction of low-abundance phosphopeptides for real complex biological samples.

  1. Separation techniques: Chromatography

    PubMed Central

    Coskun, Ozlem

    2016-01-01

    Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

  2. Fun with Paper Chromatography.

    ERIC Educational Resources Information Center

    Coleman, Dava; Hounshell, Paul B.

    1982-01-01

    Discusses paper chromatographic techniques and provides examples of typical classroom activities. Includes description of retardation values obtained during chromatography exercises and suggests using them for math lessons. (JN)

  3. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  4. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  5. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  6. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  7. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.

    PubMed

    Rudashevskaya, Elena L; Sacco, Roberto; Kratochwill, Klaus; Huber, Marie L; Gstaiger, Matthias; Superti-Furga, Giulio; Bennett, Keiryn L

    2013-01-01

    Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.

  8. Experimental and theoretical investigation of effect of spacer arm and support matrix of synthetic affinity chromatographic materials for the purification of monoclonal antibodies.

    PubMed

    Zamolo, Laura; Salvalaglio, Matteo; Cavallotti, Carlo; Galarza, Benedict; Sadler, Chris; Williams, Sharon; Hofer, Stefan; Horak, Jeannie; Lindner, Wolfgang

    2010-07-29

    The aim of this study was to elucidate the influence of each material component-the support, the spacer, and the surface chemistry-on the overall material performance of an affinity type purification media for the capture of immunoglobulin G (IgG). Material properties were investigated in terms of an experimental evaluation using affinity chromatography as well as computer modeling. The biomimetic triazine-based A2P affinity ligand was chosen as a fixed point, while spacer and support were varied. The investigated spacers were 1-2-diaminoethane (2LP), 1,3-propanedithiol (SS3), 3,6-dioxo-1,8-octanedithiol (DES), and a 1,4-substituted [1,2,3]-triazole spacer (TRZ). The support media considered were the agarose (AG) resins, PuraBead, the polyvinylether, Fractoprep, the polymethacrylate, Fractogel, and the porous silica, Fractosil. All materials were tested with pure IgG standard solution, with a mock feed solution as well as real cell culture supernatant. The interaction between IgG and A2P linked through the investigated spacers to AG was studied using molecular dynamics. The effect of a modification of the support chemical structure or of the protein-ligand binding site on the material performances was studied through target oriented simulations. Dynamic binding experiments (DBC) revealed that the performances of materials containing 2LP spacers were significantly decreased in the presence of Pluronic F68. The simulations indicated that this is probably determined by the establishment of intermolecular interactions between the 2LP charged amino group and the ether oxygen of Pluronic F68. The spacer giving the highest IgG dynamic binding capacity when Pluronic F68 was present in the feed was TRZ. The simulations showed that, among the investigated spacers, TRZ is the only one that prevents the adsorption of A2P on the support surface, thus suggesting that the mobility and lack of interaction of the ligand with the support is an important property for an affinity

  9. Liquid Chromatography in 1982.

    ERIC Educational Resources Information Center

    Freeman, David H.

    1982-01-01

    Reviews trends in liquid chromatography including apparatus, factors affecting efficient separation of a mixture (peak sharpness and speed), simplified problem-solving, adsorption, bonded phase chromatography, ion selectivity, and size exclusion. The current trend is to control chemical selectivity by the liquid phase. (Author/JN)

  10. Advanced proteomic liquid chromatography

    SciTech Connect

    Xie, Fang; Smith, Richard D.; Shen, Yufeng

    2012-10-26

    Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.

  11. Column Liquid Chromatography.

    ERIC Educational Resources Information Center

    Majors, Ronald E.; And Others

    1984-01-01

    Reviews literature covering developments of column liquid chromatography during 1982-83. Areas considered include: books and reviews; general theory; columns; instrumentation; detectors; automation and data handling; multidimensional chromatographic and column switching techniques; liquid-solid chromatography; normal bonded-phase, reversed-phase,…

  12. Low-affinity CD4+ T cells are major responders in the primary immune response

    PubMed Central

    Martinez, Ryan J.; Andargachew, Rakieb; Martinez, Hunter A.; Evavold, Brian D.

    2016-01-01

    A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5–30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion. PMID:27976744

  13. Low-affinity CD4+ T cells are major responders in the primary immune response.

    PubMed

    Martinez, Ryan J; Andargachew, Rakieb; Martinez, Hunter A; Evavold, Brian D

    2016-12-15

    A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5-30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion.

  14. RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography.

    PubMed Central

    Plevan, P; Albertini, A M; Galizzi, A; Adamoli, A; Mastromei, G; Riva, S; Cassani, G

    1977-01-01

    A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of beta factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands. Images PMID:405660

  15. Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

    PubMed

    Lintern, Katherine; Pathak, Mili; Smales, C Mark; Howland, Kevin; Rathore, Anurag; Bracewell, Daniel G

    2016-08-26

    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.

  16. Telonemia, a new protist phylum with affinity to chromist lineages

    PubMed Central

    Shalchian-Tabrizi, K; Eikrem, W; Klaveness, D; Vaulot, D; Minge, M.A; Le Gall, F; Romari, K; Throndsen, J; Botnen, A; Massana, R; Thomsen, H.A; Jakobsen, K.S

    2006-01-01

    Recent molecular investigations of marine samples taken from different environments, including tropical, temperate and polar areas, as well as deep thermal vents, have revealed an unexpectedly high diversity of protists, some of them forming deep-branching clades within important lineages, such as the alveolates and heterokonts. Using the same approach on coastal samples, we have identified a novel group of protist small subunit (SSU) rDNA sequences that do not correspond to any phylogenetic group previously identified. Comparison with other sequences obtained from cultures of heterotrophic protists showed that the environmental sequences grouped together with Telonema, a genus known since 1913 but of uncertain taxonomic affinity. Phylogenetic analyses using four genes (SSU, Hsp90, alpha-tubulin and beta-tubulin), and accounting for gamma- and covarion-distributed substitution rates, revealed Telonema as a distinct group of species branching off close to chromist lineages. Consistent with these gene trees, Telonema possesses ultrastructures revealing both the distinctness of the group and the evolutionary affinity to chromist groups. Altogether, the data suggest that Telonema constitutes a new eukaryotic phylum, here defined as Telonemia, possibly representing a key clade for the understanding of the early evolution of bikont protist groups, such as the proposed chromalveolate supergroup. PMID:16790418

  17. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  18. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.

    PubMed

    Graumann, Johannes; Dunipace, Leslie A; Seol, Jae Hong; McDonald, W Hayes; Yates, John R; Wold, Barbara J; Deshaies, Raymond J

    2004-03-01

    A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT.

  19. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency

    PubMed Central

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed. PMID:27518822

  20. Gas-phase nitronium ion affinities.

    PubMed Central

    Cacace, F; de Petris, G; Pepi, F; Angelelli, F

    1995-01-01

    Evaluation of nitronium ion-transfer equilibria, L1NO2+ + L2 = L2NO2+ + L1 (where L1 and L2 are ligands 1 and 2, respectively) by Fourier-transform ion cyclotron resonance mass spectrometry and application of the kinetic method, based on the metastable fragmentation of L1(NO2+)L2 nitronium ion-bound dimers led to a scale of relative gas-phase nitronium ion affinities. This scale, calibrated to a recent literature value for the NO2+ affinity of water, led for 18 ligands, including methanol, ammonia, representative ketones, nitriles, and nitroalkanes, to absolute NO2+ affinities, that fit a reasonably linear general correlation when plotted vs. the corresponding proton affinities (PAs). The slope of the plot depends to a certain extent on the specific nature of the ligands and, hence, the correlations between the NO2+ affinities, and the PAs of a given class of compounds display a better linearity than the general correlation and may afford a useful tool for predicting the NO2+ affinity of a molecule based on its PA. The NO2+ binding energies are considerably lower than the corresponding PAs and well below the binding energies of related polyatomic cations, such as NO+, a trend consistent with the available theoretical results on the structure and the stability of simple NO2+ complexes. The present study reports an example of extension of the kinetic method to dimers, such as L1(NO2+)L2, bound by polyatomic ions, which may considerably widen its scope. Finally, measurement of the NO2+ affinity of ammonia allowed evaluation of the otherwise inaccessible PA of the amino group of nitramide and, hence, direct experimental verification of previous theoretical estimates. PMID:11607578

  1. Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers.

    PubMed

    Hodson, B A; Pepper, D S; Dawes, J

    1991-04-19

    A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

  2. [Reliability and validity of the Japanese revised version of the television affinity scale].

    PubMed

    Erikawa, Shigeru; Yamada, Kazunari

    2012-02-01

    The purpose of this study was to improve the Japanese version of the Television Affinity Scale (TAS), and to examine the relationship between affinity for television and viewing behavior. Data was based on a random sample of 552 people in Hachioji City (Tokyo, Japan); the response rate was 55.2%. The results revealed the following: (a) the TAS 6-item version had sufficient reliability and validity, (b) the TAS provided information which could not be explained directly by demographic factors, and (c) affinity for television was positively correlated with unplanned and non-concentrated television viewing. These results are consistent with the findings of Erikawa, Yamada, Kawabata, and Numazaki (2007). In addition, the TAS scores correlated positively with entertainment program viewing. This is consistent with the findings of Rubin (1984) that television affinity correlated with ritualized television viewing. The implications of these results for contemporary television viewing are discussed.

  3. Stability of flavin semiquinones in the gas phase: the electron affinity, proton affinity, and hydrogen atom affinity of lumiflavin.

    PubMed

    Zhang, Tianlan; Papson, Kaitlin; Ochran, Richard; Ridge, Douglas P

    2013-11-07

    Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4-naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formyl-phenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol(-1). Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.

  4. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    PubMed

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-04

    The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

  5. Proton Affinity Calculations with High Level Methods.

    PubMed

    Kolboe, Stein

    2014-08-12

    Proton affinities, stretching from small reference compounds, up to the methylbenzenes and naphthalene and anthracene, have been calculated with high accuracy computational methods, viz. W1BD, G4, G3B3, CBS-QB3, and M06-2X. Computed and the currently accepted reference proton affinities are generally in excellent accord, but there are deviations. The literature value for propene appears to be 6-7 kJ/mol too high. Reported proton affinities for the methylbenzenes seem 4-5 kJ/mol too high. G4 and G3 computations generally give results in good accord with the high level W1BD. Proton affinity values computed with the CBS-QB3 scheme are too low, and the error increases with increasing molecule size, reaching nearly 10 kJ/mol for the xylenes. The functional M06-2X fails markedly for some of the small reference compounds, in particular, for CO and ketene, but calculates methylbenzene proton affinities with high accuracy.

  6. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  7. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  8. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

  9. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  10. Affine coherent states and Toeplitz operators

    NASA Astrophysics Data System (ADS)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  11. Non-affine elasticity in jammed systems

    NASA Astrophysics Data System (ADS)

    Maloney, Craig

    2006-03-01

    Symmetry dictates that perfect crystals should deform homogeneously, or affinely, under external load, and computing the elastic moduli from the underlying interaction potential is then straightforward. For disordered materials no such simple procedure exists, and recent numerical works have demonstrated that non-affine corrections can dramatically reduce the naive expectation for the shear modulus in a broad class of disordered systems and may control rigidity loss in the zero pressure limit in purely repulsive systems, i.e. the unjamming transition (c.f. [O'Hern et. al. PRE 68, 011306 (2003)]). We present numerical results and an analytical framework for the study of these non-affine corrections to the elastic response of disordered packings.

  12. Biomimetic affinity ligands for protein purification.

    PubMed

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  13. Use of Affinity Diagrams as Instructional Tools in Inclusive Classrooms.

    ERIC Educational Resources Information Center

    Haselden, Polly G.

    2003-01-01

    This article describes how the affinity diagram, a tool for gathering information and organizing it into natural groupings, can be used in inclusive classrooms. It discusses how students can be taught to use an affinity diagram, how affinity diagrams can be used to reflect many voices, and how affinity diagrams can be used to plan class projects.…

  14. Cation affinity numbers of Lewis bases.

    PubMed

    Lindner, Christoph; Tandon, Raman; Maryasin, Boris; Larionov, Evgeny; Zipse, Hendrik

    2012-01-01

    Using selected theoretical methods the affinity of a large range of Lewis bases towards model cations has been quantified. The range of model cations includes the methyl cation as the smallest carbon-centered electrophile, the benzhydryl and trityl cations as models for electrophilic substrates encountered in Lewis base-catalyzed synthetic procedures, and the acetyl cation as a substrate model for acyl-transfer reactions. Affinities towards these cationic electrophiles are complemented by data for Lewis-base addition to Michael acceptors as prototypical neutral electrophiles.

  15. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  16. On the electron affinity of B2

    SciTech Connect

    Glezakou, Vanda A.; Taylor, Peter

    2009-02-02

    We present the results of high-level ab initio calculations on the electron affinity of B2. Our new best estimate of 1.93±0.03 eV is in agreement with previous calculations as well as the sole existing experimental estimate of 1.8 eV, as derived from quantities with an uncertainty of 0.4 eV. The electron affinity of atomic boron, which is much smaller, is also calculated for comparison, and again found to be in good agreement with experiment. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  17. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  18. [Isolation and purification of enhanced green fluorescent protein using chromatography].

    PubMed

    Hou, Qinghua; Song, Shuliang; Liang, Hao; Wang, Weili; Ji, Aiguo

    2013-02-01

    Enhanced green fluorescent protein (EGFP) is a common biological marker. In this research, on the foundation of successful clone and expression of EGFP, a two-step chromatographic method was established to separate and purify EGFP, which includes the use of HisTrap HP immobilized metal affinity chromatography (IMAC) and Sephadex G-10 HR size exclusion chromatography in sequence. Sephacryl S-300 HR size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to check out the purity of EGFP. At last, it was found that EGFP still had fluorescent activity using fluorescence spectrophotometric detection and Native-PAGE detection. This method can effectively separate the active EGFP. The purity of the obtained EGFP was more than 98%.

  19. Evidence of multi-affinity in the Japanese stock market

    NASA Astrophysics Data System (ADS)

    Katsuragi, Hiroaki

    2000-04-01

    Fluctuations of the Japanese stock market (Tokyo Stock Price Index: TOPIX) are analyzed using a multi-affine analysis method. In the research to date, only some simulated self-affine models have shown multi-affinity. In most experiments using observations of self-affine fractal profiles, multi-affinity has not been found. However, we find evidence of multi-affinity in fluctuations of the Japanese stock market (TOPIX). The qth-order Hurst exponent Hq varies with changes in q. This multi-affinity indicates that there are plural mechanisms that affect the same time scale as stock market price fluctuation dynamics.

  20. The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs

    SciTech Connect

    Giannone, Richard J; Liu, Yie; Wang, Yisong

    2009-01-01

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter we describe a comprehensive methodology that utilizes our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we have demonstrated the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  1. Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

    PubMed

    Paba, Jaime; Ricart, Carlos A O; Fontes, Wagner; Santana, Jaime M; Teixeira, Antonio R L; Marchese, Jason; Williamson, Brian; Hunt, Tony; Karger, Barry L; Sousa, Marcelo V

    2004-01-01

    Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

  2. False positive RNA binding activities after Ni-affinity purification from Escherichia coli.

    PubMed

    Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinović-Carugo, Kristina; Bläsi, Udo

    2013-06-01

    A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

  3. Introduction of structural affinity handles as a tool in selective nucleic acid separations

    NASA Technical Reports Server (NTRS)

    Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

    2011-01-01

    The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

  4. Autumn Leaf Chromatography.

    ERIC Educational Resources Information Center

    Scharmann, Lawrence C.

    1984-01-01

    Describes an experiment designed to introduce students to chromatographic techniques. Also describes a teacher demonstration in which leaves obtained during the spring and fall are analyzed using chromatography. Procedures for both the experiment and the demonstration are outlined. (JN)

  5. Gas chromatography in space

    NASA Technical Reports Server (NTRS)

    Akapo, S. O.; Dimandja, J. M.; Kojiro, D. R.; Valentin, J. R.; Carle, G. C.

    1999-01-01

    Gas chromatography has proven to be a very useful analytical technique for in situ analysis of extraterrestrial environments as demonstrated by its successful operation on spacecraft missions to Mars and Venus. The technique is also one of the six scientific instruments aboard the Huygens probe to explore Titan's atmosphere and surface. A review of gas chromatography in previous space missions and some recent developments in the current environment of fiscal constraints and payload size limitations are presented.

  6. Application of chromatography technology in the separation of active components from nature derived drugs.

    PubMed

    Zhao, H-Y; Jiang, J-G

    2010-11-01

    Chromatography technology has been widely applied in various aspects of the pharmacy research on traditional Chinese medicine (TCM). This paper reviews literatures, published in the past decades, on the separation of active component from TCM using chromatography technology. Ultra-performance liquid chromatography (UPLC), high-speed counter-current chromatography (HSCCC), rapid resolution liquid chromatography (RRLC), supercritical fluid chromatography (SFC), affinity chromatography (AC), and bio-chromatography (BC) are introduced in detail. Compared to high performance of high-performance liquid chromatography (HPLC), analysis time and solvent loss are significantly reduced by UPLC with increase in resolution and sensitivity. Some ingredients from nature derived drugs can be separated more completely by HSCCC, which has remarkable characteristics such as low cost, simple operation and no pollution. Trace components from complex systems can be selectively and efficiently separated and purified by AC, This feature makes it effective in isolation and identification of active components of Chinese herbs. Interference of some impurities could be excluded by BC. Active ingredients that are difficult to be separated by normal method can be acquired by SFC. Currently, application of novel chromatography techniques in TCM is still in the exploratory stage and many problems, such as preparation of stationary phase and detection, need to be solved.

  7. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    PubMed

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  8. On modality and complexity of affine embeddings

    SciTech Connect

    Arzhantsev, I V

    2001-08-31

    Let G be a reductive algebraic group and let H be a reductive subgroup of G. The modality of a G-variety X is the largest number of the parameters in a continuous family of G-orbits in X. A precise formula for the maximum value of the modality over all affine embeddings of the homogeneous space G/H is obtained.

  9. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  10. Stabilization of the Motion of Affine Systems

    NASA Astrophysics Data System (ADS)

    Babenko, E. A.; Martynyuk, A. A.

    2016-07-01

    Sufficient conditions for the stability of a nonlinear affine system subject to interval initial conditions are established. These conditions are based on new estimates of the norms of the solutions of the systems of perturbed equations of motion. This stabilization method is used to analyze an electromechanical system with permanent magnet

  11. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  12. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  13. Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand.

    PubMed

    Baieli, María Fernanda; Urtasun, Nicolás; Miranda, María Victoria; Cascone, Osvaldo; Wolman, Federico Javier

    2014-03-01

    The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.

  14. Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity

    PubMed Central

    Kamali, Ali N.; Marín-García, Patricia; Azcárate, Isabel G.; Puyet, Antonio; Diez, Amalia; Bautista, José M.

    2015-01-01

    Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethal Plasmodium yoelii yoelii 17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses of P. yoelii yoelii 17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites. PMID:26539558

  15. Affinity-based methods in drug-target discovery.

    PubMed

    Rylova, Gabriela; Ozdian, Tomas; Varanasi, Lakshman; Soural, Miroslav; Hlavac, Jan; Holub, Dusan; Dzubak, Petr; Hajduch, Marian

    2015-01-01

    Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the "bench" scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.

  16. Ethanol increases affinity of protein kinase C for phosphatidylserine

    SciTech Connect

    Chin, J.H.

    1986-03-01

    Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition of calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.

  17. DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

    PubMed

    Forier, Cynthia; Boschetti, Egisto; Ouhammouch, Mohamed; Cibiel, Agnès; Ducongé, Frédéric; Nogré, Michel; Tellier, Michel; Bataille, Damien; Bihoreau, Nicolas; Santambien, Patrick; Chtourou, Sami; Perret, Gérald

    2017-03-17

    Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.

  18. Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor*

    PubMed Central

    Walters, Benjamin T.; Jensen, Pernille F.; Larraillet, Vincent; Lin, Kevin; Patapoff, Thomas; Schlothauer, Tilman; Rand, Kasper D.; Zhang, Jennifer

    2016-01-01

    Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors. PMID:26627822

  19. The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas.

    PubMed Central

    Velders, M. P.; van Rhijn, C. M.; Oskam, E.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1998-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism through which tumour cells, upon treatment with anti-tumour MAbs, are eliminated in vivo. However, the relative importance of various parameters that influence the efficacy of ADCC is unclear. Here we present in vitro data on the impact of MAb affinity and antigen density on ADCC, as obtained by comparison of two MAbs against the tumour-associated antigen Ep-CAM. The low-affinity MAb 17-1A (Ka = 5 x 10(7)M(-1)) currently used for therapy, and the high-affinity MAb 323/A3 (Ka = 2 x 10(9) M(-1)), were compared in ADCC experiments against murine and human tumour target cells transfected with the Ep-CAM cDNA under the control of an inducible promoter to enable regulation of the target antigen expression levels. Data obtained from these studies revealed that the high-affinity MAb, in contrast to the low-affinity MAb, could mediate killing of tumour cells with low antigen expression levels. Even at comparable MAb-binding levels, ADCC mediated by the high-affinity MAb was more effective. The kinetics of ADCC was also found to be determined by the level of antigen expression, and by the affinity and the concentration of the MAb used. The efficacy of ADCC with both low- and high-affinity MAbs further depended on adhesive interactions between effector and target cells mediated by CD18. However, at every given MAb concentration these interactions were of less importance for the high-affinity MAb than for the low-affinity MAb. As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease. PMID:9716030

  20. DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

    PubMed

    Werner, P A; Galbraith, R M; Arnaud, P

    1983-10-01

    Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

  1. Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

    PubMed

    Tomizawa, M; Latli, B; Casida, J E

    1996-10-01

    Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

  2. Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Alves, Nathan J; Bilgicer, Basar

    2013-05-21

    This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

  3. Optimal Affine-Invariant Point Matching

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Phillips, Tsaiyun I.; Shapiro, Linda G.

    1989-03-01

    The affine-transformation matching scheme proposed by Hummel and Wolfson (1988) is very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. This paper addresses the implementation of the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. It points out errors that can occur with this method due to quantization, stability, symmetry, and noise problems. By beginning with an explicit noise model which the Hummel and Wolfson technique lacks, we can derive an optimal approach which overcomes these problems. We show that results obtained with the new algorithm are clearly better than the results from the original method.

  4. Development of immobilized Sn(4+) affinity chromatography material for highly selective enrichment of phosphopeptides.

    PubMed

    Lin, Haizhu; Deng, Chunhui

    2016-11-01

    In this work, we first immobilized tin(IV) ion on polydopamine-coated magnetic graphene (magG@PDA) to synthesize Sn(4+) -immobilized magG@PDA (magG@PDA-Sn(4+) ) and successfully applied the material to highly selective enrichment of phosphopeptides. The material gathered the advantages of large surface area of graphene, superparamagnetism of Fe3 O4 , good hydrophilicity and biocompatibility of polydopamine, and strong interaction between Sn(4+) and phosphopeptides. The enrichment performance of magG@PDA-Sn(4+) toward phosphopeptides from digested β-casein at different concentrations, with and without added digested BSA was investigated and compared with magG@PDA-Ti(4+) . The results showed high selectivity and sensitivity of the Sn(4+) -IMAC material toward phosphopeptides, as good as the Ti(4+) -IMAC material. Finally, magG@PDA-Sn(4+) was applied to the analysis of endogenous phosphopeptides from a real sample, human saliva, with both MALDI-TOF MS and nano-LC-ESI-MS/MS. The results indicated that the as-synthesized Sn(4+) -IMAC material not only has good enrichment performance, but also could serve as a supplement to the Ti(4+) -IMAC material and expand the phosphopeptide coverage enriched by the single Ti(4+) -IMAC material, demonstrating the broad application prospects of magG@PDA-Sn(4+) in phosphoproteome research.

  5. Immunity in Schistosoma mansoni using antigens of Fasciola hepatica isolated by concanavalin A affinity chromatography.

    PubMed Central

    Hillyer, G V; Sagramoso de Ateca, L

    1979-01-01

    Antigens of Fasciola hepatica adult worms were chromatographed using concanavalin A-Sepharose 4B. Two unbound peaks appeared in the inclusion volume (DT-1 and DT-2), and one peak was eluted with alpha-methylglucoside (E1-1). At least seven peaks were obtained by isoelectric focusing of E1-1. The largest of these peaks, with an average pI of 4.0, contained the antigens reactive with antibodies to Schistosoma mansoni. Mice immunized with DT-2 or E1-1 and challenged with S. mansoni cercariae developed 39 to 82% fewer worms than controls. DT-1 had no protective effect. Combining DT-1 and DT-2 abolished this protection. These experiments demonstrate that F. hepatica glycoprotein antigens induce in mice significant protection to infection with S. mansoni and offer an interesting approach to the study of vaccines in experimental schistosomiasis. PMID:118932

  6. Myogenic Growth Factor Present in Skeletal Muscle is Purified by Heparin-Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Kardami, Elissavet; Spector, Dennis; Strohman, Richard C.

    1985-12-01

    A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

  7. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  8. Bicarbonate-form anion exchange: affinity, regeneration, and stoichiometry.

    PubMed

    Rokicki, Christopher A; Boyer, Treavor H

    2011-01-01

    Magnetic ion exchange (MIEX) is an effective process for removing dissolved organic carbon (DOC) from natural waters, but its implementation has been limited due to production of waste sodium chloride solution (i.e., brine) from the regeneration process. Chloride is of concern because elevated concentrations can have adverse effects on engineered and natural systems. The goal of this research was to explore the efficacy of using anion exchange resin with bicarbonate as the mobile counter ion, which would produce a non-chloride regeneration solution. It was found that bicarbonate-form MIEX resin had a similar affinity as chloride-form MIEX resin for sulfate, nitrate, DOC, and ultraviolet-absorbing substances. Both bicarbonate-form and chloride-form MIEX resins showed the greatest removal efficiencies as fresh resin, and removal efficiency decreased with multiple regeneration cycles. Nevertheless, sodium bicarbonate solution was as effective as sodium chloride solution at regenerating MIEX resin. Regeneration of the bicarbonate-form MIEX resin was illustrated by sparging carbon dioxide gas in a water/resin slurry. This regeneration process would eliminate the need for the addition of salts such as sodium chloride or sodium bicarbonate. The stoichiometry of the bicarbonate-form resin revealed that the bicarbonate was deprotonating within the resin matrix leading to a mixture of both carbonate and bicarbonate mobile counter ions. This work makes an important contribution to ion exchange applications for water treatment by evaluating the affinity, regeneration, and stoichiometry of bicarbonate-form anion exchange.

  9. Impact of crystalline quality on neuronal affinity of pristine graphene.

    PubMed

    Veliev, Farida; Briançon-Marjollet, Anne; Bouchiat, Vincent; Delacour, Cécile

    2016-04-01

    Due to its outstanding mechanical and electrical properties as well as chemical inertness, graphene has attracted a growing interest in the field of bioelectric interfacing. Herein, we investigate the suitability of pristine, i.e. without a cell adhesive coating, chemical vapor deposition (CVD) grown monolayer graphene to act as a platform for neuronal growth. We study the development of primary hippocampal neurons grown on bare graphene (transferred on glass coverslip) for up to 5 days and show that pristine graphene significantly improves the neurons adhesion and outgrowth at the early stage of culture (1-2 days in vitro). At the later development stage, neurons grown on coating free graphene (untreated with poly-L-lysine) show remarkably well developed neuritic architecture similar to those cultured on conventional poly-L-lysine coated glass coverslips. This exceptional possibility to bypass the adhesive coating allows a direct electrical contact of graphene to the cells and reveals its great potential for chronic medical implants and tissue engineering. Moreover, regarding the controversial results obtained on the neuronal affinity of pristine graphene and its ability to support neuronal growth without the need of polymer or protein coating, we found that the crystallinity of CVD grown graphene plays an important role in neuronal attachment, outgrowth and axonal specification. In particular, we show that the decreasing crystalline quality of graphene tunes the neuronal affinity from highly adhesive to fully repellent.

  10. Negative affinity X-ray photocathodes

    NASA Technical Reports Server (NTRS)

    Vanspeybroeck, L.; Kellogg, E.; Murray, S.; Duckett, S.

    1974-01-01

    A new X-ray image intensifier is described. The device should eventually have a quantum efficiency which is an order of magnitude greater than that of presently available high spatial resolution X-ray detectors, such as microchannel plates. The new intesifier is based upon a GaAs crystal photocathode which is activated to achieve negative electron affinity. Details concerning the detector concept are discussed together with the theoretical relations involved, X-ray data, and optical data.

  11. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  12. High Performance Liquid Chromatography

    NASA Astrophysics Data System (ADS)

    Talcott, Stephen

    High performance liquid chromatography (HPLC) has many applications in food chemistry. Food components that have been analyzed with HPLC include organic acids, vitamins, amino acids, sugars, nitrosamines, certain pesticides, metabolites, fatty acids, aflatoxins, pigments, and certain food additives. Unlike gas chromatography, it is not necessary for the compound being analyzed to be volatile. It is necessary, however, for the compounds to have some solubility in the mobile phase. It is important that the solubilized samples for injection be free from all particulate matter, so centrifugation and filtration are common procedures. Also, solid-phase extraction is used commonly in sample preparation to remove interfering compounds from the sample matrix prior to HPLC analysis.

  13. Micellar Electrokinetic Chromatography

    NASA Astrophysics Data System (ADS)

    Bald, Edward; Kubalczyk, Paweł

    Since the introduction of micellar electrokinetic chromatography by Terabe, several authors have paid attention to the fundamental characteristics of this separation method. In this chapter the theoretical and practical aspects of resolution optimization, as well as the effect of different separation parameters on the migration behavior are discussed. These among others include fundamentals of separation, retention factor and resolution equation, efficiency, selectivity, and various surfactants and additives. Initial conditions for method development and instrumental approaches such as mass spectrometry detection are also mentioned covering the proposals for overcoming the difficulties arising from the coupling micellar electrokinetic chromatography with mass spectrometry detection.

  14. Micellar liquid chromatography

    NASA Astrophysics Data System (ADS)

    Basova, Elena M.; Ivanov, Vadim M.; Shpigun, Oleg A.

    1999-12-01

    Background and possibilities of practical applications of micellar liquid chromatography (MLC) are considered. Various retention models in MLC, the effects of the nature and concentration of surfactants and organic modifiers, pH, temperature and ionic strength on the MLC efficiency and selectivity are discussed. The advantages and limitations of MLC are demonstrated. The performance of MLC is critically evaluated in relationship to the reversed-phase HPLC and ion-pair chromatography. The potential of application of MLC for the analysis of pharmaceuticals including that in biological fluids and separation of inorganic anions, transition metal cations, metal chelates and heteropoly compounds is described. The bibliography includes 146 references.

  15. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  16. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-08-08

    Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

  17. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  18. Vibrational photodetachment spectroscopy near the electron affinity of S2

    NASA Astrophysics Data System (ADS)

    Barrick, J. B.; Yukich, J. N.

    2016-02-01

    We have conducted laser photodetachment spectroscopy near the detachment threshold of the electron affinity of S2 in a 1.8-T field. The ions are prepared by dissociative electron attachment to carbonyl sulfide. The experiment is conducted in a Penning ion trap and with a narrow-band, tunable, Ti:sapphire laser. A hybrid model for photodetachment in an ion trap is fit to the data using the appropriate Franck-Condon factors. The observations reveal detachment from and to the first few vibrational levels of the anion and the neutral molecule, respectively. Evaporative cooling of the anion ensemble condenses the thermal distribution to the lowest initial vibrational states. The subsequent detachment spectroscopy yields results consistent with a vibrationally cooled anion population.

  19. Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts.

    PubMed

    Ahmad, Niaz; Michoux, Franck; McCarthy, James; Nixon, Peter J

    2012-04-01

    Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His₆-MBP). By using the chloroplast 16S rRNA promoter and 5' untranslated region of phage T7 gene 10, GST and His₆-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His₆-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.

  20. Chromatography in analytical biotechnology.

    PubMed

    Marchand, D H

    1994-02-01

    Recent advances in biochromatography have focused on improvements in the design of stationary supports for liquid chromatography. During the past year, the innovative use of columns packed with small non-porous particles has significantly improved the efficiency of chromatographic separations. Bioseparations that previously took hours are now possible in only a few minutes.

  1. Extending Paper Chromatography Inquiry

    ERIC Educational Resources Information Center

    Finson, Kevin

    2004-01-01

    One of the "good old" standard activities middle school students seem to enjoy is paper chromatography. The procedures and materials needed are relatively simple and the results can be colorful. All too often, the activity ends just after these colorful results are obtained, cutting short the potential it holds for some further inquiry. With some…

  2. Integrin avidity regulation: are changes in affinity and conformation underemphasized?

    PubMed

    Carman, Christopher V; Springer, Timothy A

    2003-10-01

    Integrins play critical roles in development, wound healing, immunity and cancer. Central to their function is their unique ability to modulate dynamically their adhesiveness through both affinity- and valency-based mechanisms. Recent advances have shed light on the structural basis for affinity regulation and on the signaling mechanisms responsible for both affinity and valency modes of regulation.

  3. Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity.

    PubMed

    M'Barek, Sarrah; Chagot, Benjamin; Andreotti, Nicolas; Visan, Violeta; Mansuelle, Pascal; Grissmer, Stephan; Marrakchi, Mohamed; El Ayeb, Mohamed; Sampieri, François; Darbon, Hervé; Fajloun, Ziad; De Waard, Michel; Sabatier, Jean-Marc

    2005-08-15

    Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a "gain of function" approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well-characterized maurotoxin with its variant Pi1-like disulfide bridging (MTX(Pi1)), used as a molecular template, and butantoxin (BuTX), used as an N-terminal domain provider. BuTX is found to be 60-fold less potent than MTX(Pi1) in blocking Kv1.2 (IC(50) values of 165 nM for BuTX versus 2.8 nM for MTX(Pi1)). Removal of its N-terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6-fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX-Kv1.2 interaction. Transfer of the BuTX N-terminal domain to MTX(Pi1) results in a chimera with five disulfide bridges (BuTX-MTX(Pi1)) that exhibits 22-fold greater affinity for Kv1.2 than MTX(Pi1) itself, in spite of the lower affinity of BuTX as compared to MTX(Pi1). Docking experiments performed with the 3-D structure of BuTX-MTX(Pi1) in solution, as solved by (1)H-NMR, reveal that the N-terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity.

  4. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  5. High Performance Thin Layer Chromatography.

    ERIC Educational Resources Information Center

    Costanzo, Samuel J.

    1984-01-01

    Clarifies where in the scheme of modern chromatography high performance thin layer chromatography (TLC) fits and why in some situations it is a viable alternative to gas and high performance liquid chromatography. New TLC plates, sample applications, plate development, and instrumental techniques are considered. (JN)

  6. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  7. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

  8. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  9. Protein-binding affinity of leucaena condensed tannins of differing molecular weights.

    PubMed

    Huang, Xiao Dan; Liang, Juan Boo; Tan, Hui Yin; Yahya, Rosiyah; Long, Ruijun; Ho, Yin Wan

    2011-10-12

    Depending on their source, concentration, chemical structure, and molecular weight, condensed tannins (CTs) form insoluble complexes with protein, which could lead to ruminal bypass protein, benefiting animal production. In this study, CTs from Leuceana leucocephala hybrid were fractionated into five fractions by a size exclusion chromatography procedure. The molecular weights of the CT fractions were determined using Q-TOF LC-MS, and the protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay with bovine serum albumin (BSA) as the standard protein. The calculated number-average molecular weights (M(n)) were 1348.6, 857.1, 730.1, 726.0, and 497.1, and b values (the b value represents the CT quantity that is needed to bind half of the maximum precipitable BSA) of the different molecular weight fractions were 0.381, 0.510, 0.580, 0.636, and 0.780 for fractions 1, 2, 3, 4, and 5, respectively. The results indicated that, in general, CTs of higher molecular weight fractions have stronger protein-binding affinity than those of lower molecular weights. However, the number of hydroxyl units within the structure of CT polymers also affects the protein-binding affinity.

  10. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  11. Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

    2014-09-07

    In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

  12. Characterization of receptor proteins using affinity cross-linking with biotinylated ligands.

    PubMed

    Shinya, Tomonori; Osada, Tomohiko; Desaki, Yoshitake; Hatamoto, Masahiro; Yamanaka, Yuko; Hirano, Hisashi; Takai, Ryota; Che, Fang-Sik; Kaku, Hanae; Shibuya, Naoto

    2010-02-01

    The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

  13. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  14. Identification of Integrin β Subunit Mutations That Alter Affinity for Extracellular Matrix Ligand*

    PubMed Central

    Kendall, Timmy; Mukai, Leona; Jannuzi, Alison L.; Bunch, Thomas A.

    2011-01-01

    We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells. PMID:21757698

  15. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1.

  16. High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface.

    PubMed

    Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W; Linhardt, Robert J; Sharp, Joshua S

    2015-04-24

    Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.

  17. High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface*

    PubMed Central

    Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W.; Linhardt, Robert J.; Sharp, Joshua S.

    2015-01-01

    Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes. PMID:25752613

  18. Latest European coelacanth shows Gondwanan affinities.

    PubMed

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  19. On the electron affinity of Be2

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Partridge, H.

    1984-01-01

    Calculations of the electron affinity (EA) of Be2 using a large Slater-type orbital basis set and extensive correlation based upon a CASSCF reference are reported. The adiabatic EAs are estimated to be 0.44 eV for the 2Sigma sub g(+) state and 0.56 eV for the 2Pi sub u state. The extra electron attaches into an empty bonding orbital, causing a shortening of the bond length and an increase in omega(e). The D(e) of the 2Pi sub u state of Be2 is six times as large as the D(e) of Be2.

  20. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  1. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    PubMed

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

  2. HLA class I alleles are associated with peptide-binding repertoires of different size, affinity, and immunogenicity.

    PubMed

    Paul, Sinu; Weiskopf, Daniela; Angelo, Michael A; Sidney, John; Peters, Bjoern; Sette, Alessandro

    2013-12-15

    Prediction of HLA binding affinity is widely used to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (percentage) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of ~30,000 dengue virus-derived peptides only 0.3% were predicted to bind HLA A*0101, whereas nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms used, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702), or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigated the genetic and structural basis of the phenomenon. Although no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than are HLA A alleles.

  3. Age-dependent decrease in the affinity of muscarinic M1 receptors in neocortex of rhesus monkeys.

    PubMed Central

    Vannucchi, M G; Goldman-Rakic, P S

    1991-01-01

    In vitro autoradiography on tissue sections and receptor assay in cortical membrane homogenates revealed that pirenzepine high-affinity muscarinic sites (M1) decrease in affinity in the prefrontal cortex and in other cortical areas of aged rhesus monkey (Macaca mulatta). Carbachol competition experiments detected only a single, low-affinity class of sites in old monkeys, while two classes of sites (low and high affinity) were observed in young adults. The change in affinity in the aged monkeys is not accompanied by a decrease in the density of these sites and, further, the age-related decline in the affinity of the M1 site is reversible. In the presence of Mg2+, the M1 muscarinic receptors in the aged monkeys were capable of forming carbachol high-affinity sites. These results provide evidence for age-dependent functional changes in receptor activity in cerebral cortex and indicate that these receptors maintain a degree of plasticity that could be a strategic target for research aimed at treatment of memory disorders in aged humans. Images PMID:1763062

  4. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  5. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  6. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  7. Chromatography in Industry

    NASA Astrophysics Data System (ADS)

    Schoenmakers, Peter

    2009-07-01

    This review focuses on the chromatography research that has been carried out within industry or in close cooperation with industry and that has been reported in the scientific literature between 2006 and mid-2008. Companies in the health care sector, such as pharmaceutical and biotechnology companies, are the largest contributors. Industrial research seems to take place in an open environment in cooperation with academia, peer companies, and institutions. Industry appears ready to embrace new technologies as they emerge, but they focus strongly on making chromatography work robustly, reliably, rapidly, and automatically. “Hyphenated” systems that incorporate on-line sample-preparation techniques and mass-spectrometric detection are the rule rather than the exception. Various multidimensional separation methods are finding numerous applications. Strategies aimed at speeding up the development of new chromatographic methods remain the focus of attention. Also, there is a clear trend toward exploring chromatographic methods for parallel processing along with other strategies for high-throughput analysis.

  8. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  9. Hydrazide-functionalized affinity on conventional support materials for glycopeptide enrichment.

    PubMed

    Sajid, Muhammad Salman; Jabeen, Fahmida; Hussain, Dilshad; Ashiq, Muhammad Naeem; Najam-Ul-Haq, Muhammad

    2017-02-24

    In affinity chromatography, enrichment of biomolecules is dependent on the selection of affinity sites immobilized onto a suitable support material. A few hydrazide - functionalized materials with surface modification protocols compatible to conventional support materials like silica and cellulose are reported. The study demonstrates the modification/derivatization pathways that can be adopted to modify the support materials with similar surface chemistry like cellulose, poly(GMA/DVB), or diamond. Poly(GMA/DVB) and cellulose represent hydrophilic supports whereas diamond is a hydrophobic support material. SEM images of three materials provide surface morphology whereas FT-IR confirms reaction completion and derivatization. These hydrazide - functionalized materials are applied to fetuin digest for glycopeptides enrichment and subsequently for selectivity and sensitivity assessment. Statistically, poly(GMA/DVB) shows 85.7% sensitivity with specificity of 88.8% in the enrichment experiments. Diamond offers hydrophobic interactions to non-glycopeptides and they co-elute with glycopeptides, resulting in reduced sensitivity down to 69.2%. Poly(GMA/DVB) shows recovery up to 89%, while recovery for cellulose and diamond is 83 and 71%, respectively. The materials enrich mono-N-linked-glycosylated peptide from tryptic digest of chicken avidin spiked in fetuin digest. The hydrazide group density on cellulose, poly(GMA/DVB), and diamond is 2.8, 2.3, and 2.1 mmol/g, respectively; this contributes towards the specificity and sensitivity of designed materials. The materials are also applied to serum samples and enriched glycopeptides characteristic of serum glycoproteins of clinical importance. Therefore this study provides routes for the economical surface modifications of support materials and to fabricate affinity materials with improved efficiency. Graphical Abstract Glycopeptides enrichment by hydrazine affinity.

  10. Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography.

    PubMed

    Kölln, Johanna; Braren, Ingke; Bredehorst, Reinhard; Spillner, Edzard

    2007-01-01

    The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.

  11. Strategy for affinity maturation of an antibody with high evolvability to (4-hydroxy-3-nitrophenyl) acetyl hapten.

    PubMed

    Furukawa, Koji; Shimizu, Takeyuki; Murakami, Akikazu; Kono, Ryo; Nakagawa, Masatoshi; Sagawa, Takuma; Yamato, Ichiro; Azuma, Takachika

    2007-03-01

    In order to quantitate the contribution of amino acid replacements to an increase in affinity during affinity maturation, we measured thermodynamic parameters of the antigen-antibody interaction for a group of anti-(4-hydroxy-3-nitrophenyl) acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. We prepared a common ancestor and hypothetical intermediate clones that might occur on the affinity maturation pathway, by employing site-directed mutagenesis. Isothermal calorimetric titration of the antigen-antibody reaction revealed that antibody evolution proceeds in two steps. The first step is driven by a decrease in enthalpy, in which two amino acid replacements in the VL region play an essential role. Further accumulation of amino acid replacements in VH and VL regions during the second step induce a progressive increase in affinity, which is driven by an increase in entropy, which has a cooperative mutational effect.

  12. An automatic system for multidimensional integrated protein chromatography.

    PubMed

    Kong, Yingjun; Li, Xiunan; Bai, Gaoying; Ma, Guanghui; Su, Zhiguo

    2010-10-29

    An automatic system for multidimensional integrated protein chromatography was designed for simultaneous separation of multiple proteins from complex mixtures, such as human plasma and tissue lysates. This computer-controlled system integrates several chromatographic columns that work independently or cooperatively with one another to achieve efficient high throughputs. The pipelines can be automatically switched either to another column or to a collection container for each UV-detected elution fraction. Environmental contamination is avoided due to the closed fluid paths and elimination of manual column change. This novel system was successfully used for simultaneous preparation of five proteins from the precipitate of human plasma fraction IV (fraction IV). The system involved gel filtration, ion exchange, hydrophobic interaction, and heparin affinity chromatography. Human serum albumin (HSA), transferrin (Tf), antithrombin-III (AT-III), alpha 1-antitrypsin (α1-AT), and haptoglobin (Hp) were purified within 3 h. The following recovery and purity were achieved: 95% (RSD, 2.8%) and 95% for HSA, 80% (RSD, 2.0%) and 99% for Tf, 70% (RSD, 2.1%) and 99% for AT-III, 65% (RSD, 2.0%) and 94% for α1-AT, and 50% (RSD, 1.0%) and 90% for Hp. The results demonstrate that this novel multidimensional integrated chromatography system is capable of simultaneously separating multiple protein products from the same raw material with high yield and purity and it has the potential for a wide range of multi-step chromatography separation processes.

  13. Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

    PubMed Central

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  14. Mepyramine-JNJ7777120-hybrid compounds show high affinity to hH(1)R, but low affinity to hH(4)R.

    PubMed

    Wagner, Eva; Wittmann, Hans-Joachim; Elz, Sigurd; Strasser, Andrea

    2011-11-01

    In literature, a synergism between histamine H(1) and H(4) receptor is discussed. Furthermore, it was shown, that the combined application of mepyramine, a H(1) antagonist and JNJ7777120, a H(4) receptor ligand leads to a synergistic effect in the acute murine asthma model. Thus, the aim of this study was to develop new hybrid ligands, containing one H(1) and one H(4) pharmacophor, connected by an appropriate spacer, in order to address both, H(1)R and H(4)R. Within this study, we synthesized nine hybrid compounds, which were pharmacologically characterized at hH(1)R and hH(4)R. The new compounds revealed (high) affinity to hH(1)R, but showed only low affinity to hH(4)R. Additionally, we performed molecular dynamic studies for some selected compounds at hH(1)R, in order to obtain information about the binding mode of these compounds on molecular level.

  15. The affine cohomology spaces and its applications

    NASA Astrophysics Data System (ADS)

    Fraj, Nizar Ben; Laraiedh, Ismail

    2016-12-01

    We compute the nth cohomology space of the affine Lie superalgebra 𝔞𝔣𝔣(1) on the (1,1)-dimensional real superspace with coefficient in a large class of 𝔞𝔣𝔣(1)-modules M. We apply our results to the module of weight densities and the module of linear differential operators acting on a superspace of weighted densities. This work is the generalization of a result by Basdouri et al. [The linear 𝔞𝔣𝔣(n|1)-invariant differential operators on weighted densities on the superspace ℝ1|n and 𝔞𝔣𝔣(n|1)-relative cohomology, Int. J. Geom. Meth. Mod. Phys. 10 (2013), Article ID: 1320004, 9 pp.

  16. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field.

  17. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  18. Immunoaffinity centrifugal precipitation chromatography.

    PubMed

    Qi, Lin; Ito, Yoichiro

    2007-06-01

    Purification of proteins based on immunoaffinity has been performed using a solid support coated with antibody against the target proteins. The method requires immobilizing the antibody onto the solid support using protein A or G, and has a risk of adsorptive loss of target proteins onto the solid support. Centrifugal precipitation chromatography has been successfully used to purify enzymes, such as ketosteroid isomerase and hyaluronidase without the use of solid support. The purpose of this study is to demonstrate that immunoaffinity centrifugal precipitation chromatography is capable of isolating an antigen by exploiting antigen-antibody binding. The separation was initiated by filling both channels with 40% saturated ammonium sulfate (AS) of pH 4-4.5 followed by loading 20 microl of human plasma (National Institutes of Health blood bank) mixed with 2 mg of rabbit anti-HSA (human serum protein) antibody (Sigma). Then, the sample channel was eluted with water at 0.03 ml/min and AS channel with 40% AS solution of pH 4-4.5 at 1 ml/min until all non-binding components were eluted. Then, the releasing reagent (50% AS solution containing 0.5 M glycine and 10% ammonium hydroxide at pH 10) was introduced through the AS channel to release the target protein (HSA). The retained antibody was recovered by eluting the sample channel with water at 1 ml/min. A hollow fiber membrane device at the outlet (MicroKros, Spectrum, New Brunswick, NJ, USA) was provided on-line dialysis of the eluent before fractions were collected, so that the fractions could be analyzed by SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) without further dialysis. The current method does not require immobilizing the antibody onto a matrix, which is used by the conventional immunoaffinity chromatography. This method ensures full recovery of the antigen and antibody, and it may be applied to purification of other proteins.

  19. Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-06-01

    The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

  20. Principles of Liquid Chromatography

    NASA Astrophysics Data System (ADS)

    Bakalyar, Stephen R.

    This article reviews the basic principles of high performance liquid chromatography (HPLC). The introductory section provides an overview of the HPLC technique, placing it in historical context and discussing the elementary facts of the separation mechanism. The next section discusses the nature of resolution, describing the two principal aspects, zone center separation and zone spreading. The third section takes a detailed look at how HPLC is used in practice to achieve a separation. It discusses the three key variables that need to be adjusted: retention, efficiency, and selectivity. A fourth section is concerned with various relationships of practical importance: flow rate, temperature, and pressure. A final section discusses future trends in HPLC.

  1. Affinity partitioning of restriction endonucleases. Application to the purification of EcoR I and EcoR V.

    PubMed

    Vlatakis, G; Bouriotis, V

    1991-02-01

    Partitioning of restriction endonucleases between two liquid aqueous phases can be strongly influenced by group-specific ligands included in the two-phase system. Three restriction endonucleases, namely EcoR I, EcoR V and BamH I, were partitioned within an aqueous dextran-polyethylene glycol (PEG) system. The enzymes could be extracted into the upper PEG phase by using either triazine dyes or herring DNA as affinity ligands. The influence of the endogenous bacterial nucleic acids, concentration of polymerbound dye and concentration of sodium chloride on the system were examined. A partial purification of EcoR I (up to 52-fold) and EcoR V (up to 37-fold) was achieved using a combination of affinity partitioning and ion-exchange chromatography, providing an extremely fast and economical method for the isolation of restriction endonucleases free from contaminating nuclease activities.

  2. Smooth affine shear tight frames: digitization and applications

    NASA Astrophysics Data System (ADS)

    Zhuang, Xiaosheng

    2015-08-01

    In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

  3. Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.

    PubMed

    Yi, Lian; Wang, Xue; Bethge, Lucas; Klussmann, Sven; Roper, Michael G

    2016-03-21

    The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

  4. Spiral Countercurrent Chromatography

    PubMed Central

    Ito, Yoichiro; Knight, Martha; Finn, Thomas M.

    2013-01-01

    For many years, high-speed countercurrent chromatography conducted in open tubing coils has been widely used for the separation of natural and synthetic compounds. In this method, the retention of the stationary phase is solely provided by the Archimedean screw effect by rotating the coiled column in the centrifugal force field. However, the system fails to retain enough of the stationary phase for polar solvent systems such as the aqueous–aqueous polymer phase systems. To address this problem, the geometry of the coiled channel was modified to a spiral configuration so that the system could utilize the radially acting centrifugal force. This successfully improved the retention of the stationary phase. Two different types of spiral columns were fabricated: the spiral disk assembly, made by stacking multiple plastic disks with single or four interwoven spiral channels connected in series, and the spiral tube assembly, made by inserting the tetrafluoroethylene tubing into a spiral frame (spiral tube support). The capabilities of these column assemblies were successfully demonstrated by separations of peptides and proteins with polar two-phase solvent systems whose stationary phases had not been well retained in the earlier multilayer coil separation column for high-speed countercurrent chromatography. PMID:23833207

  5. Investigating the Affinities and Persistence of VX Nerve Agent in Environmental Matrices

    SciTech Connect

    Love, A H; Vance, A L; Reynolds, J G; Davisson, M L

    2004-03-09

    Laboratory experiments were conducted to determine environmental variables that affect the affinities and persistence of the nerve agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) at dilute concentrations in environmental matrices. Quantitative analyses of VX and its degradation products were performed using LC-MS. Batch hydrolysis experiments demonstrated an increasing hydrolysis rate as pH increased, as shown in previous studies, but also indicated that dissolved aqueous constituents can cause significant differences in the absolute hydrolysis rate. Adsorption isotherms from batch aqueous experiments revealed that VX has a high affinity for hydrophobic organics, a moderate affinity for montmorillonite clay, and a very low affinity for an iron-oxyhydroxide soil mineral, goethite. The adsorption on goethite was increased with the presence of dissolved organic matter in solution. VX degraded rapidly when dried onto goethite, when an inner-sphere complex was forced. No enhanced degradation occurred with goethite in small amounts water. These results suggest that aqueous conditions have important controls on VX adsorption and degradation in the environment and a more mechanistic understanding of these controls is needed in order to enable accurate predictions of its long-term fate and persistence.

  6. Investigating the affinities and persistence of VX nerve agent in environmental matrices.

    PubMed

    Love, Adam H; Vance, Andrew L; Reynolds, John G; Davisson, M Lee

    2004-12-01

    Laboratory experiments were conducted to determine environmental variables that affect the affinities and persistence of the nerve agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) at dilute concentrations in environmental matrices. Quantitative analyses of VX and its degradation products were performed using LC-MS. Batch hydrolysis experiments demonstrated an increasing hydrolysis rate as pH increased, as shown in previous studies, but also indicated that dissolved aqueous constituents can cause significant differences in the absolute hydrolysis rate. Adsorption isotherms from batch aqueous experiments revealed that VX has a high affinity for hydrophobic organics, a moderate affinity for montmorillonite clay, and a very low affinity for an iron-oxyhydroxide soil mineral, goethite. The adsorption on goethite was increased with the presence of dissolved organic matter in solution. VX degraded rapidly when dried onto goethite, when specific adsorption was forced. No enhanced degradation occurred with goethite in small amounts of water. These results suggest that aqueous conditions have important controls on VX adsorption and degradation in the environment and a more mechanistic understanding of these controls is needed in order to enable accurate predictions of its long-term fate and persistence.

  7. Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering.

    PubMed

    Thakkar, Shraddha; Nanaware-Kharade, Nisha; Celikel, Reha; Peterson, Eric C; Varughese, Kottayil I

    2014-01-14

    Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP.

  8. Identification of a Small Molecule that Increases Hemoglobin Oxygen Affinity and Reduces SS Erythrocyte Sickling

    PubMed Central

    2015-01-01

    Small molecules that increase the oxygen affinity of human hemoglobin may reduce sickling of red blood cells in patients with sickle cell disease. We screened 38 700 compounds using small molecule microarrays and identified 427 molecules that bind to hemoglobin. We developed a high-throughput assay for evaluating the ability of the 427 small molecules to modulate the oxygen affinity of hemoglobin. We identified a novel allosteric effector of hemoglobin, di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1). TD-1 induced a greater increase in oxygen affinity of human hemoglobin in solution and in red blood cells than did 5-hydroxymethyl-2-furfural (5-HMF), N-ethylmaleimide (NEM), or diformamidine disulfide. The three-dimensional structure of hemoglobin complexed with TD-1 revealed that monomeric units of TD-1 bound covalently to β-Cys93 and β-Cys112, as well as noncovalently to the central water cavity of the hemoglobin tetramer. The binding of TD-1 to hemoglobin stabilized the relaxed state (R3-state) of hemoglobin. TD-1 increased the oxygen affinity of sickle hemoglobin and inhibited in vitro hypoxia-induced sickling of red blood cells in patients with sickle cell disease without causing hemolysis. Our study indicates that TD-1 represents a novel lead molecule for the treatment of patients with sickle cell disease. PMID:25061917

  9. Studies toward bivalent κ opioids derived from salvinorin A: heteromethylation of the furan ring reduces affinity.

    PubMed

    Munro, Thomas A; Xu, Wei; Ho, Douglas M; Liu-Chen, Lee-Yuan; Cohen, Bruce M

    2013-12-20

    The recent crystal structure of the κ-opioid receptor (κ-OR) revealed, unexpectedly, that the antagonist JDTic is a bivalent ligand: in addition to the orthosteric pocket occupied by morphinans, JDTic also occupies a distinct (allotopic) pocket. Mutagenesis data suggest that salvinorin A (1) also binds to this allotopic pocket, adjacent to the aspartate residue that anchors the basic nitrogen atom of classical opiates (Asp138). It has been suggested that an H-bond donor appended to 1 might interact with Asp138, increasing affinity. Such a bivalent ligand might also possess altered functional selectivity. Based on modeling and known N-furanylmethyl opioid antagonists, we appended H-bond donors to the furan ring of 1. (Dimethylamino)methyl groups at C-15 or C-16 abolished affinity for κ-OR. Hydroxymethylation at C-16 was tolerated, but 15,16-bis-hydroxymethylation was not. Since allosteric modulators may go undetected in binding assays, we also tested these and other low-affinity derivatives of 1 for allosteric modulation of dynorphin A in the [(35)S]GTPγS assay. No modulation was detected. As an alternative attachment point for bivalent derivatives, we prepared the 2-(hydroxyethoxy)methyl ether, which retained high affinity for κ-OR. We discuss alternative design strategies for linked, fused or merged bivalent derivatives of 1.

  10. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation

    PubMed Central

    Kepler, Thomas B.; Munshaw, Supriya; Wiehe, Kevin; Zhang, Ruijun; Yu, Jae-Sung; Woods, Christopher W.; Denny, Thomas N.; Tomaras, Georgia D.; Alam, S. Munir; Moody, M. Anthony; Kelsoe, Garnett; Liao, Hua-Xin; Haynes, Barton F.

    2014-01-01

    Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain–light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases. PMID:24795717

  11. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation.

    PubMed

    Kepler, Thomas B; Munshaw, Supriya; Wiehe, Kevin; Zhang, Ruijun; Yu, Jae-Sung; Woods, Christopher W; Denny, Thomas N; Tomaras, Georgia D; Alam, S Munir; Moody, M Anthony; Kelsoe, Garnett; Liao, Hua-Xin; Haynes, Barton F

    2014-01-01

    Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain-light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases.

  12. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these th