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Dissertations / Theses on the topic 'Metal ion binding'
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1
Satofuka, Hiroyuki. "Studies on heavy metal ion-binding peptides : Application for heavy metal ion detection and detoxification." 京都大学 (Kyoto University), 2002. http://hdl.handle.net/2433/149818.
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Sekaly, Amina L. R. "Trace metal ion binding to fulvic acids in model systems and freshwaters." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/NQ57625.pdf.
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Sekaly, Amina L. R. (Amina Lula R. ). Carleton University Dissertation Chemistry. "Trace metal ion binding to fulvic acids in model systems and freshwaters." Ottawa, 2000.
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Li, Zheng. "Structural Studies of Natural and Synthetic Macromolecules Stabilized by Metal Ion Binding." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1295646060.
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Prasannan, Charulata Bhaskaran. "Modulation of restriction enzyme PvuII activity by metal ion cofactors." Diss., St. Louis, Mo. : University of Missouri--St. Louis, 2009. http://etd.umsl.edu/r4461.
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Jeong, Chang-Yoon. "Modelling metal competition for adsorption sites on humic acid." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389363.
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Buckelew, Aurelie Lina. "Investigation of metal-ion binding in the four-way junction construct of the hairpin ribozyme." Texas A&M University, 2003. http://hdl.handle.net/1969.1/2307.
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The hairpin ribozyme is a small catalytic RNA that cleaves a phosphodiester
bond. In order for cleavage to occur, the hairpin ribozyme must properly fold into its
docked conformation, in which the two loops interact to form the active site. Metal ions
and the four-way junction play critical roles in the stabilization of the docked
conformation. The work presented in this thesis attempts to investigate the metal-ion
dependence of the docking of the four-way junction construct of the hairpin ribozyme. In
addition, the activity of the hairpin ribozyme in the presence of Mn2+ was observed.
Initially, a four-stranded four-way junction construct of the hairpin ribozyme and a
loopless mutant were characterized by native gel electrophoresis and thermal
denaturation to verify ribozyme formation.
A novel interaction between the sulfur of a phosphorothioate-substituted
mononucleotide, such as adenosine thiomonophosphate (AMPS) or adenosine
thiotrisphoshate (ATPgS), and Cd2+ has been characterized by UV-vis spectroscopy. A
feature at 208 nm was identified to be a result of sulfur-to-Cd2+ transfer. The apparent
binding affinities, the apparent extinction coefficients, and the binding ratios were
determined for each complex.
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Reyzer, Michelle Lee. "Evaluation of metal binding interactions in host-guest chemistry using quadrupole ion trap mass spectrometry /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004364.
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Burkitt, William Ian. "Metal-binding non-covalent protein complexes studied by electrospray ionisation and Fourier transform ion cyclotron resonance mass spectrometry." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429814.
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Zhang, Ting. "Lipid Speciation and Ion Interactions at the Air-Aqueous Interface in Atmospheric Aerosol Model Systems." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu152416015716577.
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Zhang, Zhiling. "The structure and function of troponin T upon metal ion binding and the detection of nucleic acid sequence variations." Thesis, University of North Texas, 2005. https://digital.library.unt.edu/ark:/67531/metadc5565/.
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Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.
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Erat, Michèle Christine. "Two domains of branching and catalysis act as specific metal ion binding platforms within the group II intron ribozyme core /." Zürich, 2007. http://opac.nebis.ch/cgi-bin/showAbstract.pl?u20=9783952338100.
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Roy, Choudhury Shamali. "Mapping of metal ion binding sites in the RecBCD enzyme and the role of magnesium in the subunit interactions of RecBCD." College Park, Md. : University of Maryland, 2006. http://hdl.handle.net/1903/3360.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2006.Thesis research directed by: Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Attah, Isaac Kwame. "BINDING ENERGIES AND SOLVATION OF ORGANIC MOLECULAR IONS, REACTIONS OF TRANSITION METAL IONS WITH, AND PLASMA DISCHARGE IONIZATION OF MOLECULAR CLUSTERS." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/525.
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In this dissertation, different approaches have been employed to address the quest of understanding the formation and growth mechanisms of carbon-containing molecular ions with relevance to astrochemistry. Ion mobility mass spectrometry and DFT computations were used to investigate how a second nitrogen in the pyrimidine ring will affect the formation of a covalent bond between the benzene radical cation and the neutral pyrimidine molecule, after it was shown that a stable covalent adduct can be formed between benzene radical cation and the neutral pyridine. Evidence for the formation of a more stable covalent adduct between the benzene radical cation and the pyrimidine is reported here. The effect of substituents on substituted-benzene cations on their solvation by an HCN solvent was also investigated using ion mobility mass spectrometry and DFT computations were also investigated. We looked at the effect of the presence of electron-withdrawing substituents in fluorobenzene, 1,4 di- fluorobenzene, and benzonitrile on their solvation by up to four HCN ligands, and compared it to previous work done to determine the solvation chemistry of benzene and phenylacetylene by HCN. We report here the observed increase in the binding of the HCN molecule to the aromatic ring as the electronegativity of the substituent increased. We also show in this dissertation, DFT calculations that reveal the formation of both hydrogen-bonded and electrostatic isomers, of similar energies for each addition to the ions respectively. The catalytic activity of the 1st and 2nd row TM ions towards the polymerization of acetylene done using the reflectron time of flight mass spectrometry and DFT calculations is also reported in this dissertation. We explain the variation in the observed trend in C-H/C-C activity of these ions. We also report the formation of carbide complexes by Zr+, Nb+, and Mo+, with the acetylene ligands, and show the thermodynamic considerations that influence the formation of these dehydrogenated ion-ligand complexes. Finally, we show in this dissertation, a novel ionization technique that we employed to generate ions that could be relevant to the interstellar and circumstellar media using the reflectron time of flight mass spectrometry.
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Lappalainen, K. (Katja). "Modification of native and waste starch by depolymerization and cationization:utilization of modified starch in binding of heavy metal ions from an aqueous solution." Doctoral thesis, Oulun yliopisto, 2015. http://urn.fi/urn:isbn:9789526209661.
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Abstract Starch is one of the most abundant polysaccharides found in nature and is widely utilized in various fields of industry. Due to the complex structure of native starch it is insoluble in most organic solvents and needs modification prior utilization. In this study, ionic liquids (ILs), modern green chemistry alternatives for common solvents were used as reaction media in starch modification. At first various starch species were depolymerized in 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) with p-TsOH as a catalyst. Microwave activation or conventional bath heating were used as heating methods while HPLC-ELSD was used as an analytical method. All studied starch species depolymerized similarly into water-soluble starch oligomers while microwave activation shortened the depolymerization time considerably compared to oil bath heating. Barley starch was chosen for further experiments, in which various ILs were studied as potential media for starch dissolution and depolymerization. Results suggested that both the anion and the cation part of the IL had an effect on the dissolution and depolymerization of barley starch. After the depolymerization reactions, the depolymerized barley starch was further modified by cationization. [AMIM]Cl was used as the reaction media, microwave activation as the heating method while HPLC-ELSD, 1H NMR and elemental analysis were used as analytical methods. The modified products had DS values from 0.2 to 0.5 depending on the reaction conditions. The products were studied as potential binding agents for heavy metal ions which showed that moderately substituted modified starch (DS 0.4) could be used to bind Cu(II), Fe(III) and Zn(II) ions from an aqueous solution. Finally, potato peel waste was studied as an alternative starch source to produce cationized starch for wastewater purification. Peel waste was pre-treated by alkaline depolymerization after which it was cationized in a water solution to produce cationized products with DS from 0 to 0.35. The cationized peel waste products were studied preliminary as binding agents for Cu(II) ions from a water solution using ICP-OES as an analytical method. The results suggested that when the molar ratio between cationized waste starch and copper was 3:1, cationized waste starch was an effective binding agent for copper ionsTiivistelmä Tärkkelys on yksi yleisimmistä luonnossa esiintyvistä polysakkarideista. Sitä hyödynnetään useilla eri teollisuuden aloilla. Monimutkaisen rakenteensa vuoksi tärkkelys on liukenematon useimpiin orgaanisiin liuottimiin ja veteen, minkä vuoksi sitä täytyy modifioida ennen käyttöä. Tässä väitöstutkimuksessa tärkkelyksen modifioinnissa käytettiin ionisia nesteitä reaktioväliaineena. Tutkimuksen alussa eri tärkkelyslajeja depolymeroitiin 1-allyyli-3-metyyli-imidatsoliumkloridissa ([AMIM]Cl) katalyyttinä p-TsOH. Mikroaaltoaktivointia ja haudekuumennusta käytettiin vaihtoehtoisina lämmitysmenetelminä. Reaktion edistymistä ja tuotteiden muodostumista tutkittiin HPLC-ELSD -menetelmällä. Eri tärkkelyslajit depolymeroituivat samankaltaisesti vesiliukoisiksi, lyhytketjuisiksi tärkkelysoligomeereiksi. Mikroaaltoaktivointi lyhensi reaktioaikaa haudekuumennukseen verrattuna. Tutkimuksen seuraavassa vaiheessa tutkittiin ohratärkkelyksen liukoisuutta ja depolymeroitumista eri ionisissa nesteissä. Tulosten perusteella ionisen nesteen sekä anioni- että kationiosa vaikuttivat tärkkelyksen liukenemiseen. Depolymeroidun ohratärkkelyksen modifiointitutkimuksia jatkettiin [AMIM]Cl:ssa kationisoinnilla. Lämmitysmenetelmänä käytettiin mikroaaltoaktivointia. Tuotteet tutkittiin käyttäen alkuaineanalyysiä sekä HPLC-ELSD- että 1H NMR-tekniikoita. Kationisoitujen tuotteiden substituutioaste (DS) vaihteli reaktio-olosuhteista riippuen välillä 0.2–0.5. Saatuja tuotteita tutkittiin raskasmetalli-ionien sitomisessa vesiliuoksesta. Havaittiin, että kohtalaisesti substituoitu (DS 0.4) modifioitu tärkkelys sitoi Cu(II)-, Fe(III)- ja Zn(II)-ioneja vesiliuoksesta. Tutkimuksen loppuosassa tutkittiin perunan kuorijätettä vaihtoehtoisena tärkkelyslähteenä kationisoidun tärkkelyksen valmistamisessa. Kuorijäte esikäsiteltiin kuumentamalla se emäksisessä etanoliliuoksessa, minkä jälkeen sille suoritettiin kationisointi vesiliuoksessa. Kationisten tuotteiden substituutioasteet vaihtelivat välillä 0–0.35. Tuotteiden soveltuvuutta Cu(II)-ionien sitomiseen vesiliuoksesta tutkittiin ICP-OES -menetelmän avulla. Alustavien tulosten mukaan kationisoitu jätetärkkelys sitoi kupari-ioneja vedestä, kun tärkkelyksen ja kuparin moolisuhde oli 3:1
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Weidenbach, Stevi. "INVESTIGATION OF THE MECHANISM OF ACTION FOR MITHRAMYCIN AND THE BIOSYNTHESIS OF L-REDNOSE IN SAQUAYAMYCINS." UKnowledge, 2017. http://uknowledge.uky.edu/pharmacy_etds/77.
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Natural products continue to be a major chemical lead matter for drug discovery due to their diverse chemical structures and bioactivities. Clinically significant natural products include anti-cancer and anti-infective compounds and while many more of these compounds show promising bioactivity, their clinical relevance is often limited by toxicity or poor solubility. Combinatorial biosynthesis can be employed to modify existing chemical scaffolds towards reducing these limitations. To fully take advantage of these biochemical tools, it is important to understand the biosynthesis and mechanism of action of the molecules.
Saccharides in glycosylated natural products provide specific interactions with cellular targets and are often crucial for a compound’s bioactivity. Genetic engineering of sugar pathways can modify glycosylation patterns leading to the diversification of natural products. Saquayamycins (SQN) H and I are cytotoxic angucycline antibiotics containing five deoxyhexoses including the rare amino sugar rednose. Elucidating the biosynthetic pathway of rednose could add to the arsenal of combinatorial biosynthesis tools for drug development. Our research goal of investigating the rednose biosynthetic pathway was pursued through two specific aims: the identification of the Streptomyces sp. KY 40-1 gene cluster involved in the biosynthesis of SQN H and I (sqn) (specific aim 1), and the validation of the proposed L-rednose biosynthetic pathway up to the glycosyl transfer through enzymatic synthesis of NDP-3,6-dideoxy-L-idosamine (specific aim 2). The sqn gene cluster revealed deoxysugar biosynthetic genes that could be used to alter glycosylation patterns to generate novel compounds while the enzymatic synthesis afforded novel genetic engineering tools to generate novel TDP-deoxysugars that could be used to diversify compounds such as aminoglycosides to circumvent resistance mechanisms. The first step to generate TDP-glucosamine enzymatically was accomplished, however later steps were unsuccessful.
The aureolic acid mithramycin (MTM) was recently tested in clinical trials for Ewing sarcoma following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 present only in Ewing sarcoma cells It is understood that MTM binds the minor groove of G/C rich DNA as an Mg2+-coordinated dimer disrupting transcription of proto-oncogenes; however, the DNA recognition rules were not completely understood, making further interrogation of MTM’s DNA binding preferences necessary. This research goal of further understanding the mechanism of action for MTM was approached through two specific aims: the investigation of the dimerization of MTM (specific aim 3), and the investigation of MTM’s DNA binding preferences (specific aim 4). This work established that MTM and its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK), mithramycin SA tryptophan (MTM SA-Trp), and mithramycin SA alanine (MTM SA-Ala) dimerize even in the absence of DNA under physiologically relevant conditions. The study also demonstrated that modification of the 3-side chain modulates DNA binding affinity of MTM analogues, established a minimum MTM binding site on DNA, and revealed MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout laying the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.
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Suzuki, Noriaki. "Applications of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and x-ray photoelectron spectroscopy (XPS) to study interactions of genetically engineered proteins with noble metal films /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/10618.
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Shahir, Shafinaz. "Engineering and the maltose binding protein for metal ions sensing." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434921.
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Alghamdi, Ebtehal. "DFT Study on the Binding of Selected Metal Ions with Phenylalanine Dipeptide." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2019. http://digitalcommons.auctr.edu/cauetds/181.
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In this study, M06-2X/6-311+G(2d,2p) level calculations were performed to examine the binding energies and vibrational frequencies of different conformers of phenylalanine dipeptide interacting with metal ions (Na+, K+, Mg2+ and Ca2+). Four conformers were selected from the list of 20 most stable structures. The main goal was to understand the influence of conformers on the binding affinity of metal ions with different conformers of phenylalanine dipeptide. In agreement with experimental results, interactions of metal ions with two aromatic rings along with lone pair electrons of oxygen produced high stability. Binding energy was lowest for the metal ion interacting with only one aromatic ring. This study revealed the binding affinity order of metal ions Mg2+ > Ca2+ > Na+ > K+ with any of the conformers considered for phenylalanine dipeptide.
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Keech, Angus Miles. "Role of cobalt(II) and manganese(II) as optical and magnetic probes of metal binding sites in proteins." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389267.
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Serkiz, Steven Michael. "Factors affecting the binding of protons and metal ions to naturally occurring dissolved organic matter." Diss., Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/25799.
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Muehl, Brian S. "The synthesis and study of phosphine crown ether ligands, and an investigation of how the binding of sodium or potassium ions affects the donor ability of the phosphorus center." Virtual Press, 1992. http://liblink.bsu.edu/uhtbin/catkey/834529.
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The phosphine crown ether, 16-(4'diphenylphosphinophenyl)-1,4,7,10,13-pentaoxa-16azacyclooctadecane (III), was synthesized using a reaction scheme beginning with n-phenyldiethanolamine and the dichloride of tetraethylene glycol, with an overall yield of 4%. Platinum and Palladium complexes of the ligand, of the form MC12L2, were synthesized as well. 13C NMR and picrate extraction data indicate III and IV (the crown-5 analog) both moderately bind sodium (14%, 15%) and potassium ions (17%, 28%). Compound V (a crown-5, triphenylphosphine-based ligand) will bind both sodium and potassium ions as well (18%, 6%). When IV is complexed to nickel carbonyl (Ni(CO)3), the addition of sodium and potassium ions cause the Al carbonyl stretching frequency to increase slightly (0.3 cm-1, 0.2 cm 1). For comparison, the addition of a proton causes the A1 carbonyl stretching frequency to increase 5.2 cm-1. However, the shift in the A1 carbonyl stretching frequency upon the addition of sodium or potassium ions indicates that ion binding by the crown ether is communicated to the phosphorus and finally to the carbonyl groups.Ball State UniversityMuncie, IN 47306Department of Chemistry
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Yang, Ying. "Mechanism of metal delivery and binding to transport sites of Cu+-transporting ATPases." Link to electronic thesis, 2005. http://www.wpi.edu/Pubs/ETD/Available/etd-042905-112044/.
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Grady, Amanda Ellen. "The regulation of the type 5 cyclic nucleotide phosphodiesterase in airway smooth muscle by metal ions and small molecular weight proteins." Thesis, University of Strathclyde, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366810.
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Schweitzer, Dirk. "Biomimetic models of the active site of the metalloenzyme nitrile hydratase /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8692.
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Hribesh, Samira. ""2'-deoxy-6-thioguanosine : synthesis of monomer, oligomers and long DNA, and their binding with metal ions." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/2353.
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The formation of one-dimensional (1D) coordination polymers using thiolated DNA nucleosides and nucleotides with transition metals was investigated. 2’-Deoxy-6- 1 13 thioguanosine was successfully synthesised and characterized by H-NMR, C-NMR, IR, MS and UV spectroscopy. Subsequently, the binding of several transition metal ions to 2’-deoxy-6-thioguanosine and 6-thioguanosine was studied. The binding ratio of thio-monomer with metal ions was shown to be three ligands to one metal in the case of cobalt (II) and nickel (II), whilst in the case of the cadmium (II)-complex with the thio-monomer, the results suggested that a 1D coordination polymer might be formed, although no crystal data was obtained to confirm this structure. Therefore, the oligonucleotides containing the thiolated monomer were synthesised to extend this study. The preparation of thiolated homo-guanosine oligomers involved standard solid phase synthesis of oligo-guanosine followed by on-column conversion of guanosine to 6-thioguanosine. The thiolated reaction was the same as that used for the monomer and yielded 2-, 3-, 4- and 5-mer thio-oligomers in good yield. The thio-oligomers were purified by HPLC and were fully characterized by mass spectrometry and UV. The interaction of thio-oligomers with cadmium (II) ions was investigated. For the dimer and tetramer, the binding ratio was two thio-bases bound to one cadmium, whereas for the trimer and pentamer the binding ratio showed more than one binding species in solution. In order to synthesise a long thiolated DNA polymer, an enzymatic slippage reaction was performed, where a short primer-template of poly (dG)-poly (dC) was 1010 - extended up to 300 bp using Klenow exo polymerase and nucleosides triphosphate (d-tGTP and d-CTP). The addition of cadmium ions to thio-modified DNA suggested a structure resembling M-DNA as coordination polymer is obtained according to UV titration data.
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Shyy, Yeun-Jund. "Nuclear magnetic resonance studies on the interaction of metal ions with adenine nucleotides and substrates binding to adenylate kinase /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487329662147312.
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Al, Bittar Sheiraz. "3-Deoxyanthocyanins : Chemical synthesis, structural transformations, affinity for metal ions and serum albumin, antioxidant activity." Thesis, Avignon, 2016. http://www.theses.fr/2016AVIG0264.
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Ce travail porte sur la synthèse chimique d’analogues simples d’anthocyanes, une classe majeure de pigments naturels solubles dans l’eau. Onze ions flavylium substitués par des groupements hydroxyl,méthoxyl et beta-D-glucopyranosyloxyl en positions 4’, 5 et 7 ont été préparés en utilisant des procédures simples. De plus, les deux principales 3-désoxyanthocyanidines du sorgho rouge, l’apigéninidine (APN) et la lutéolinidine (LTN), ont été synthétisées en une seule étape. Les propriétés physico-chimiques ainsi que l’activité antioxydante ont été étudiées pour le chlorure de 3’,4’,7-trihydroxyflavylium (P1), son 7-O-beta-D-glucoside (P2) et le chlorure de 3’,4’,5,7-tétrahydroxyflavylium (LTN). Grâce à leur noyau catéchol, ces pigments complexent rapidement FeIII, AlIII and CuI et ne se lient que faiblement à FeII tout en stimulant son autoxydation en FeIII. Suite à la complexation de CuII, les pigments subissent une oxydation. Les aglycones P1 et LTN sont des ligands modérés de l’albumine de sérum humain (HSA) et leurs chalcones ont montré une plus grande affinité pour la HSA que leurs formes colorées. Leur capacité antioxydante a été démontrée par le test de réduction du radical stable DPPH et par l’inhibition de la peroxydation lipidique induite par le fer héminique, un modèle de stress oxydant postprandial dans l’estomac. Les aglycones P1 et LTN (particulièrement, dans leur forme incolore chalcone) sont plus efficaces que le glucoside P2This work deals with the chemical synthesis of simple analogs of anthocyanins, the main class of watersolublenatural pigments. Eleven flavylium ions with hydroxyl, methoxyl and beta-D-glucopyranosyloxylsubstituents at positions 4’, 5 and 7 have been prepared by straightforward chemical procedures.Moreover, the two main 3-deoxyanthocyanidins of red sorghum, apigeninidin (APN) and luteolinidin(LTN), have been synthesized in a one-step protocol. The physicochemical properties and antioxidantactivity are investigated for 3’,4’,7-trihydroxyflavylium chloride (P1), its 7-O-beta-D-glucoside (P2) and3’,4’,5,7-tetrahydroxyflavylium chloride (LTN). Owing to their catechol B-ring, they rapidly bind FeIII,AlIII and CuI, more weakly interact with FeII while promoting its autoxidation to FeIII. Following CuIIbinding, the pigments undergo oxidation. Aglycones P1 and LTN are moderate ligands of human serumalbumin (HSA) with chalcones having a higher affinity for HSA than the corresponding colored forms.The antioxidant activity of P1, P2 and LTN is investigated via two tests: reduction of the stable DPPHradical and inhibition of heme-induced lipid peroxidation (a model of postprandial oxidative stress inthe stomach). Aglycones P1 and LTN (especially in their colorless chalcone form) are more potent thanglucoside P2
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Elison, Kalman Grim. "Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea." Thesis, Uppsala universitet, Strukturbiologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-387943.
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This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR.
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Hoang, Huy Ngoc. "Metal clips for folding peptides : a study of palladium (II) binding to histidine residues in short peptides stabilize (sic) their a-Helical conformation in solutions /." St. Lucia, Qld, 2003. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17478.pdf.
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Li, Weijia, and n/a. "Development of New Binding Phases for Speciation Measurements of Trace Metals with the Diffusive Gradients in Thin Films Technique." Griffith University. School of Environmental and Applied Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040504.150905.
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The recently developed technique of diffusive gradients in thin films (DGT) for speciation measurement of analytes in the environment was further developed through the development of series of new binding phases including poly(acrylamide-co-acrylic acid) copolymer hydrogel (PAM-PAA), poly(acrylamidoglycolic acid-co-acrylamide) (PAAGA-PAM) hydrogel, the Whatman P81 cellulose phosphate ion exchange membrane (P81) and a liquid binding phase of poly(4-styrenesulfonate) (PSS). A new diffusion layer, cellulose dialysis membrane, was also employed for the liquid binding phase DGT. PAM-PAA copolymer hydrogel was prepared by the controlled hydrolysis of polyacrylamide (PAM) in an alkaline solution of 10% sodium hydroxide. The capacity of the copolymer hydrogel to bind various metal ions was tested under a range of uptake conditions. Ions such as Cu2+ and Cd2+ were bound more strongly to the copolymer hydrogel than the competing ions such as Na+, K+, Ca2+ and Mg2+. Metals bound to the copolymer hydrogel can be efficiently eluted in 2 M HNO3 solution (>94%). Application of this new binding material to DGT technique was validated in a synthetic lake water (Windermere, Lake District, UK) with a recovery of 99.0% for Cu2+. PAAGA-PAM hydrogel was prepared by copolymerising 2-acrylamidoglycolic acid with acrylamide. The metal ion binding properties of the hydrogel were characterised for Cu2+, Cd2+ and competing ions under various experimental conditions. The hydrogel was shown to bind Cu2+ and Cd2+ strongly under non-competitive binding conditions, with binding capacities of 5.3 and 5.1 micromol cm-2, respectively. The binding capacity of each metal decreased, under competitive binding conditions (with a range of metal ions present at 17.8 mN), to 1.3 and 0.17 micromol cm-2, respectively, indicating a strong selective binding towards Cu2+. The metal ions were readily recovered (>94%) by eluting with 2 M HNO3. Finally, the copolymer hydrogel was tested as a binding phase with the DGT technique. A linear mass vs. time relationship was observed for Cu2+ in Windermere water with a recovery of close to 100%. The use of a commercially available solid ion exchange membrane (P81) as the binding phase in DGT analysis was demonstrated. P81 is a strong cation exchange membrane. Its performance characteristics as a new binding phase in DGT measurement of Cu2+ and Cd2+ were systematically investigated. Several advantages over the conventional ion exchange resin-embedded hydrogel based binding phases used in DGT were observed. These include: simple preparation, ease of handling, and reusability. The binding phase preferentially binds to transition metal ions rather than competing ions. Within the optimum pH range (pH 4.0 - 9.0), the maximum non-competitive binding capacities of the membrane for Cu2+ and Cd2+ were 3.22 and 3.07 micromol cm-2, respectively. The suitability of the new membrane-based binding phase for DGT applications was validated experimentally. The results demonstrated excellent agreement with theoretically predicted trends. The reusability of this binding phase was also investigated. Application of a liquid binding phase and a dialysis membrane diffusive layer were proposed for the first time. The binding phase was a 0.020 M solution of poly(4-styrenesulfonate) (PSS) polyelectrolyte using a specially designed DGT device. The binding properties of Cd2+, Cu2+, and a range of alkali and alkaline earth metal ions to the PSS solution were characterised. The PSS behaved like a cation exchanger with preferential binding to Cd2+ (6.0 micromole ml-1, log K = 9.0) and Cu2+ (2.5 micromole ml-1, log K = 8.1) under competitive binding conditions. The DGT devices were successfully validated for Cd2+ and Cu2+ in Windermere water. The speciation performance of the solid and liquid binding phases developed in this study was investigated in solutions containing ethylenediaminetetraacetic acid disodium salt (EDTA), humic acid (HA), glucose (GL), dodecylbenzenesulfonic acid (DBS) and tannic acid (TA) with Cu2+ and Cd2+. The ratios of labile metals over total metals were at good agreement with calculated theoretical values using Stability Constants Database. The results indicated that the DGT-labile concentration measured by DGT with these binding phases is essentially free metal ion concentration in the sample. All newly developed DGT binding phases were successfully applied for environmental speciation. The field deployments were carried out in both freshwater and salt-water test sites.
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Riihimäki, Eva-Stina. "Structure and Dynamics of the Copper-binding Octapeptide Region in the Human Prion Protein." Licentiate thesis, KTH, Chemistry, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-250.
Full textAbstract:
The copper-binding ability of the prion protein may be closely connected to its function. Identifying the exact function of the prion protein can clarify the underlying mechanism in prion diseases. In this work, the copper-binding octapeptide region in the human prion protein has been studied. The structural characteristics of the binding site are examined by quantum chemical structural optimization. The calculations aim at identifying a substitute for copper(II) to be used in NMR-spectroscopic studies of the copper-binding region. The dynamical and structural features of the peptide region are investigated in molecular dynamics simulations. Aspects of importance in the development of model systems in molecular dynamics simulation are addressed.
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Seifert, Steffen. "Synthese und Komplexbildungseigenschaften ausgewählter Maillard-Reaktionsprodukte." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1232923513056-87374.
Full textAbstract:
Zahlreiche Studien belegen, dass Maillard-Reaktionsprodukte (MRP) in vivo einen Einfluss auf den physiologischen Metallionenhaushalt haben können. Da bisher noch keine Korrelation zwischen dem Entstehen von definierten MRP und einem erhöhten Metallionenbindungsvermögen ermittelt werden konnte, war es das Ziel dieser Arbeit, die Komplexbildungseigenschaften der ausgewählten MRP Nε-Carboxymethyllysin, Isomaltol und Maltosin sowie deren Strukturanaloga Maltol, Deferipron, Mimosin und Pyridosin mit den physiologisch relevanten Metallionen Cu(II), Zn(II), Fe(III), Al(III) und Mn(II) zu untersuchen. Dazu wurden die MRP Nε-Carboxymethyllysin und Maltosin sowie die parallel untersuchten Substanzen Pyridosin, Maltosin-3-benzylether, Nα-Hippuryl- und Nα-Acetylmaltosin in ausreichender Menge und Reinheit synthetisiert. Dabei gelang es, für Maltosin und Pyridosin neue und effiziente Synthesewege zu entwickeln, bei welchen zum ersten Mal beide Substanzen gezielt aufgebaut wurden. Die Komplexbildungskonstanten der Liganden mit den Metallionen wurden pH-potentiometrisch bestimmt (I[KNO3] = 0,15 M; θ = 25 °C). Durch die Auswertung der Protonierungskonstanten der gebildeten Komplexe und das Vermessen geeigneter Derivate konnten für die untersuchten Komplexe zusätzlich die Koordinationsstellen der Liganden aufgeklärt werden. Die Untersuchungen zu den Komplexbildungseigenschaften bestätigten erstmals die Vermutung, dass MRP in der Lage sind, Metallionen zu binden. Dabei wurde weiterhin ermittelt, dass die Bindung von Cu(II) durch Nε-Carboxymethyllysin und von Fe(III), Al(III) und Cu(II) durch Maltosin durchaus von physiologischer Relevanz sind. Die Bedeutung der Ergebnisse wurde qualitativ durch Versuche mit Maltosin-derivatisiertem Rinderserumalbumin unterstrichen. Als besonderes Ergebnis der Arbeit ist herauszustellen, dass das MRP Maltosin und die Verbindung Pyridosin deutlich stabilere Komplexe mit Fe(III) bilden als das zur Fe(III)-Chelattherapie eingesetzte Medikament Deferipron. Diese festgestellte Eigenschaft bietet interessante Perspektiven für zukünftige Studien zur möglichen Anwendung von z. B. Maltosin als PharmakonSeveral studies show that Maillard reaction products (MRP) may influence the physiological metal ion balance. But none of these studies prove a correlation between the formation of defined MRP and an enhanced metal ion binding. Therefore it was the aim of this work to investigate the complex formation characteristics of the selected MRP Nε-carboxymethyllysine, isomaltol and maltosine as well as the structural analogues maltol, deferiprone, mimosine and pyridosine with the physiological relevant metal ions Cu(II), Zn(II), Fe(III), Al(III) and Mn(II). For that purpose the MRP Nε-carboxymethyllysine and maltosine plus the parallel analysed substances pyridosine, maltosine-3-benzylether, Nα-hippuryl- and Nα-acetylmaltosine were synthesised. Thereby new and efficient syntheses for maltosine and pyridosine were developed. The stability constants of the ligands with the metal ions were determined by pH-potentiometry (I(KNO3) = 0,15 M; θ = 25 °C). Furthermore the donor atoms within the formed complexes were determined by the evaluation of the protonation constants of the formed complexes and by the analysis of adequate derivatives. The studies to the complex formation characteristics confirm for the first time the assumption, that MRP are able to form stable complexes with metal ions. Withal it was ascertained that the coordination of Cu(II) by Nε-carboxymethyllysine and of Fe(III), Al(III) and Cu(II) by maltosine may be of physiological relevance. The significance of the results was pointed out by experiments with maltosine derivatised bovine serum albumine. The fact that the MRP maltosine and the compound pyridosine form more stable complexes with Fe(III) as the medicament for the Fe(III) chelate therapy deferiprone is a particular result of this work. This property affords interesting perspectives for future studies about a possible appliance of e.g. maltosine as pharmaceutical
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Seifert, Steffen. "Synthese und Komplexbildungseigenschaften ausgewählter Maillard-Reaktionsprodukte." Doctoral thesis, Technische Universität Dresden, 2008. https://tud.qucosa.de/id/qucosa%3A23758.
Full textAbstract:
Zahlreiche Studien belegen, dass Maillard-Reaktionsprodukte (MRP) in vivo einen Einfluss auf den physiologischen Metallionenhaushalt haben können. Da bisher noch keine Korrelation zwischen dem Entstehen von definierten MRP und einem erhöhten Metallionenbindungsvermögen ermittelt werden konnte, war es das Ziel dieser Arbeit, die Komplexbildungseigenschaften der ausgewählten MRP Nε-Carboxymethyllysin, Isomaltol und Maltosin sowie deren Strukturanaloga Maltol, Deferipron, Mimosin und Pyridosin mit den physiologisch relevanten Metallionen Cu(II), Zn(II), Fe(III), Al(III) und Mn(II) zu untersuchen. Dazu wurden die MRP Nε-Carboxymethyllysin und Maltosin sowie die parallel untersuchten Substanzen Pyridosin, Maltosin-3-benzylether, Nα-Hippuryl- und Nα-Acetylmaltosin in ausreichender Menge und Reinheit synthetisiert. Dabei gelang es, für Maltosin und Pyridosin neue und effiziente Synthesewege zu entwickeln, bei welchen zum ersten Mal beide Substanzen gezielt aufgebaut wurden. Die Komplexbildungskonstanten der Liganden mit den Metallionen wurden pH-potentiometrisch bestimmt (I[KNO3] = 0,15 M; θ = 25 °C). Durch die Auswertung der Protonierungskonstanten der gebildeten Komplexe und das Vermessen geeigneter Derivate konnten für die untersuchten Komplexe zusätzlich die Koordinationsstellen der Liganden aufgeklärt werden. Die Untersuchungen zu den Komplexbildungseigenschaften bestätigten erstmals die Vermutung, dass MRP in der Lage sind, Metallionen zu binden. Dabei wurde weiterhin ermittelt, dass die Bindung von Cu(II) durch Nε-Carboxymethyllysin und von Fe(III), Al(III) und Cu(II) durch Maltosin durchaus von physiologischer Relevanz sind. Die Bedeutung der Ergebnisse wurde qualitativ durch Versuche mit Maltosin-derivatisiertem Rinderserumalbumin unterstrichen. Als besonderes Ergebnis der Arbeit ist herauszustellen, dass das MRP Maltosin und die Verbindung Pyridosin deutlich stabilere Komplexe mit Fe(III) bilden als das zur Fe(III)-Chelattherapie eingesetzte Medikament Deferipron. Diese festgestellte Eigenschaft bietet interessante Perspektiven für zukünftige Studien zur möglichen Anwendung von z. B. Maltosin als Pharmakon.Several studies show that Maillard reaction products (MRP) may influence the physiological metal ion balance. But none of these studies prove a correlation between the formation of defined MRP and an enhanced metal ion binding. Therefore it was the aim of this work to investigate the complex formation characteristics of the selected MRP Nε-carboxymethyllysine, isomaltol and maltosine as well as the structural analogues maltol, deferiprone, mimosine and pyridosine with the physiological relevant metal ions Cu(II), Zn(II), Fe(III), Al(III) and Mn(II). For that purpose the MRP Nε-carboxymethyllysine and maltosine plus the parallel analysed substances pyridosine, maltosine-3-benzylether, Nα-hippuryl- and Nα-acetylmaltosine were synthesised. Thereby new and efficient syntheses for maltosine and pyridosine were developed. The stability constants of the ligands with the metal ions were determined by pH-potentiometry (I(KNO3) = 0,15 M; θ = 25 °C). Furthermore the donor atoms within the formed complexes were determined by the evaluation of the protonation constants of the formed complexes and by the analysis of adequate derivatives. The studies to the complex formation characteristics confirm for the first time the assumption, that MRP are able to form stable complexes with metal ions. Withal it was ascertained that the coordination of Cu(II) by Nε-carboxymethyllysine and of Fe(III), Al(III) and Cu(II) by maltosine may be of physiological relevance. The significance of the results was pointed out by experiments with maltosine derivatised bovine serum albumine. The fact that the MRP maltosine and the compound pyridosine form more stable complexes with Fe(III) as the medicament for the Fe(III) chelate therapy deferiprone is a particular result of this work. This property affords interesting perspectives for future studies about a possible appliance of e.g. maltosine as pharmaceutical.
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Li, Jyn-Han, and 李居翰. "MIB: a Metal Ion–Binding sites prediction tool." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/8rn739.
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Graichen, Adam. "Enhanced detection strategies accomplished through metal binding and miniature mass spectrometry." 2013. https://scholarworks.umass.edu/dissertations/AAI3556252.
Full textAbstract:
A multiplexed method for performing MS/MS on multiple ions simultaneously in a miniature rectilinear ion trap (RIT) mass spectrometer has been developed. This method uses an ion encoding procedure that relies on the mass bias that exists when ions are externally injected into an RIT operated with only a single phase RF applied to one pair of electrodes. The ion injection profile under such conditions ions is Gaussian-like over a wide range of RF amplitudes, or low mass cutoff (LMCO) values, during ion accumulation. We show that this distribution is related to ion m/z, and is likely caused by ions having an optimal range of pseudo-potential well depths for efficient trapping. Based on this observation, precursor ion intensity changes between two different injection LMCO values can be predicted, and these ion intensity changes are found to be carried through to their corresponding product ions, enabling multiplexed MS/MS spectra to be deconvoluted. The gas-phase reactions of a series of coordinatively unsaturated [Ni(L) n]y+ complexes, where L is a nitrogen-containing ligand, with chemical warfare agent (CWA) simulants in a miniature rectilinear ion trap mass spectrometer were investigated as part of a new approach to detect CWA. Results show that the metal complex ions can react with low concentrations of several CWA simulants, including dipropyl sulfide (simulant for mustard gas), acetonitrile (simulant for the nerve agent tabun), and diethyl phosphite (simulant for nerve agents sarin, soman, tabun, and VX), thereby providing a sensitive means of detecting these compounds. The [Ni(L)n] 2+ complexes are found to be particularly reactive with the simulants of mustard gas and tabun, allowing their detection at low parts-per-billion (ppb) levels. These detection limits are well below the median lethal doses for these CWAs, which indicates the applicability of this new approach, and are about two orders of magnitude lower than electron ionization detection limits on the same mass spectrometer. The use of coordinatively unsaturated metal complexes as reagent ions offers the possibility of further tuning the ion-molecule chemistry so that desired compounds can be detected selectively or at even lower concentrations. Mass spectrometry has become a tool for studying noncovalently bound complexes. Specifically, electrospray ionization mass spectrometry (ESI-MS) has found increasing use for the determination of affinity (Ka) or dissociation (Kd) constants. Direct measurement of the equilibrium components by ESI-MS is the most straightforward approach for determining binding equilibrium constants, but this approach is prone to error and has some inherent limitations. Transferring complexes from solution to the gas phase may perturb the equilibrium concentrations and/or different ionization efficiencies may cause the resulting ion signals not to reflect actual solution concentrations. Furthermore, ESI only works under a limited range of solvent conditions (i.e. low ionic strengths), which limits the broad applicability of this approach. We propose an approach based on covalent labeling in the context of metal-catalyzed oxidation (MCO) reactions that, when combined with MS, overcomes such limitations when determining metal-ligand binding constants. The MCO-MS approach will provide concurrent information regarding metal binding site and metal-protein binding affinity. Optimization of the MCO reaction through isotopic mass tags will permit enhanced identification of modified residues. Application of this method to study the affinity and binding interactions of other divalent metals with β2m are likely to provide insight into the specificity of copper for causing β2m amyloid formation.
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Cates, Mary Susan. "Crystallographic and computational studies of the metal ion binding properties of parvalbumin." Thesis, 2000. http://hdl.handle.net/1911/19477.
Full textAbstract:
An astonishing number of important physiological processes are regulated by the small alkaline earth metal, calcium. Regulatory Ca2+-binding proteins must be able to distinguish Ca2+ ions in the presence of greater concentrations of other metal cations, such as Mg2+, Na+ and K+. The EF-hand family is a large class of Ca2+-binding proteins that displays this sort of preferential Ca2+-binding. The secondary and tertiary structure of the EF-hand metal ion binding site is highly conserved from one member of the family to the next. Because of this conservation, we can use the small, amenable, EF-hand protein, parvalbumin, as a model system to study the mechanisms that define the metal ion affinities and specificities of EF-hand Ca2+-binding sites in general.
Our collaborator, Dr. James Potter, has designed a mutant to test directly the role of the last coordinating residue in the EF-hand binding site, the PVEF-E101D parvalbumin mutant. The crystal structures of both the Ca 2+- and Mg2+-bound complexes of PVEF-E101D have been determined. The PVEF-E101D mutant displayed a 100-fold decrease in the binding affinity for Ca2+, and the Mg2+-binding affinity was increased 10-fold. Moreover, the Ca2+ off-rate escalated from 1 s--1 in wild-type parvalbumin to 600 s--1 in the PVEF-E101D mutant. The conformation of the mutated EF-hand in the PVEF-E101D/Mg2+ structure was typical of a Mg 2+-bound EF-hand, with the exception of an F helix movement of ∼1 A toward the bound cation that allowed the shorter aspartate residue to coordinate the Mg2+ ion. The PVEF-E101D/Ca2+ structure showed that the aspartate residue is unable to bind Ca2+ in the bidentate mode normally adopted by the wild type glutamate. The resulting sixfold Ca2+ coordination in the mutant is usually characteristic of Mg2+-bound EF-hands, and this finding indicates that the binding loop is not sufficiently flexible to allow the aspartate residue to move in far enough to offer bidentate ligation of the Ca2+ ion.
Two MD simulations were used to further investigate the relationship between the last coordinating residue of the EF-hand binding loop and the overall plasticity and flexibility of the loop region. The first simulation, called Alchemy, simulated the transition from Ca2+ to Mg 2+ coordination through varying the van der Waals parameters for the bound metal ions. The glutamate at position 12 was accurately and reversibly predicted to be the source of bidentate ligation of Ca2+ in our simulations. A second simulation, the Aspartate simulation, produced results that correlated well with the experimental result that an E101D substitution at EF loop position 12 resulted in monodentate Ca2+ coordination. The F helix was able to move in to the binding cavity during the simulation to allow one aspartate oxygen to bind the Ca2+ ion, but the aspartate was unable to achieve a favorable orientation for bidentate Ca 2+ coordination. The findings indicate that the interplay between the last coordinating residue of the loop, and the plasticity, or flexibility, of the binding loop, to a great extent determines the species of cations that are allowed to bind in a particular EF-hand site.
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Wu, Keng-Fu, and 伍耕甫. "Synthesis and Metal Ion Binding Behavior of 1,8-Bisisoxazole-9H-Carbazole Derivatives." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/78692641891320292002.
Full textAbstract:
碩士國立中山大學
化學系研究所
102
Carbazole based fluorescence chemosensor 23, 1,8-bis(3-phenyl-isoxazolyl)-3,6- di-tert-butyl-9H-carbazole, was prepared and characterized by NMR, MALDI-MS, absorption and emission spectroscopy. The sensing properties of 23 toward various metal ions were investigated in CH3CN solution. A highly selective fluorescent turn-off response is observed in the presence of copper (II) among other metal ions, as is observed with the addition of 10 equiv of Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Cd3+, Mn2+, Ni2+, Ag+, Pb2+, Zn2+, Cu2+, Hg2+ ions. The fluorescence intensity is almost not influenced by the addition of other metal ions. A Job’s plot determining the stoichiometry ratio of 23 and Cu2+ in CH3CN indicates a simple one-to-one binding preference. The binding constant for Cu2+ ion with 23 is 2.2 × 104 M-1. Another carbazole based chemosensor 24 is derived from 23. We found that 24 can bind to six metal ions. Especially with nickel ions and zinc ions, significant color change can be easily observed by naked eyes.
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Yang, Te-Chien, and 楊得謙. "Application of bacterial heavy metal ion binding proteins for silver resistance and adsorption." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/18326510462067964364.
Full textAbstract:
碩士國立中興大學
生命科學院碩士在職專班
97
Heavy metal pollution has posed one of the serious threat to human health, therefore, seeks an economy and effective method to clear the heavy metal pollution is the urgent matter.Bioremediation is the best method can clear heavy metal pollution completely and will not cause the second pollution. Because the bacterial heavy metal binding proteins can absorb the heavy metal ions, therefore we can use the biological characteristics to eliminate the heavy metal pollution in the soil or water. Take the SilE protein as the example, the SilE protein has played the key role in the resistant mechanism of silver resistant bacterium, therefore, the Ag+ adsorptive capacity of SilE protein will be closely linked to its silver resistance. Six strains were included in this project. E.coli strain J53 contains plasmid pMG101 which have silver resistance. There are nine open reading frames of the silver resistance determinant in the plasmid, including SilE protein. Two recombinant plasmids were constructed by cloning silE gene with or without signal sequence into pET21b vector, resulting SilE-SP and SilE-NSP respectively. For MerP protein, another two constructs were made by cloning MerP with signal sequence or B95P without signal sequence into pET21b vector. All recombinant plasmids were transformed to E.coli BL21 (DE3) strain. One control is with backbone pET21b in E.coli BL21 (DE3). Resistance tests in agar plate and in liquid medium were performed to detect the strength of resistance to Ag+. In disc assay, the strength was pMG101＞SilE-SP＞MerP＞pET21b＞SilE-NSP＞B95P and pMG101＞MerP＞SilE-SP＞pET21b＞SilE-NSP＞B95P for resistance test in liquid medium. Because both SilE and MerP protein can absorb heavy metal ions, adsobability experiment was also done to detect the Ag+ residue in the buffer. The results were 1.89 ± 0.026 mg/l for pMG101，1.92 ± 0.031 mg/l for MerP，1.93 ± 0.006 mg/l for pET21b，1.94 ± 0.021 mg/l for SilE-SP，1.94 ± 0.020 mg/l for SilE-NSP，and 2.10 ± 0.026 mg/l for B95P. In brief, the adsorbability to adsorb Ag+ is pMG101＞MerP＞pET21b＞SilE-SP≒SilE-NSP＞B95P. The results above showed that MerP protein can absorb Ag＋ and the efficiency is only less than pMG101（J53）, therefore, the next experiment was to observe the growth and detect the amount of the Ag＋ can be absorbed by the transgenic plant containing merP gene （P5 transgenic plant）. The host of bacterial merP gene is Arabidopsis. Furthermore, because MerP protein can absorb Hg+2, Ag+ and other heavy metals, the character of adsorption to heavy metal ions needs to be further investigated for the P5 transgenic plant. We found the differences for silver adsorption of P5 transgenic plant embedding in MS medium containing between Ag+ only and both Hg+2 and Ag+.The results indicated that the plant can absorb more Ag+ in the condition containing both Hg+2 and Ag+.
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Oxenford, Leah R. "Characterization of N-isopropyl acrylamide based polymers for pH sensing and metal ion binding." 2006. http://digital.library.okstate.edu/etd/umi-okstate-2091.pdf.
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Davis, Jared Henry. "Structure, metal ion binding, and dynamics of the GAAA tetraloop-receptor interaction in solution." 2007. http://www.library.wisc.edu/databases/connect/dissertations.html.
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Ausenhus, Scott L. "Phosphoenolpyruvate carboxylase from maize function of the divalent metal ion in binding and catalysis /." 1993. http://catalog.hathitrust.org/api/volumes/oclc/31386451.html.
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Sui, Meng-Jiun, and 隋孟君. "Characterization of the divalent metal ion and the phosphate ion binding sites in the H-N-H endonulease colicin E7." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/56562691591829371051.
Full textAbstract:
博士國防醫學院
生命科學研究所
91
Colicins are a group of plasmid-encoded protein toxins produced by Escherichia coli to kill other sensitive E. coli and closely related coliform bacteria. The cytotoxic activities of colicins against their target cells are well documented: as an RNase, a DNase, a protein synthesis inhibitor or an ionophore. The subfamily of E-group DNase-type colicins, including colicin E2, E7, E8 and E9, contain an H-N-H motif in the cytotoxic nuclease domain, capable of cleaving DNA nonspecifically. This H-N-H motif was firstly identified in a subfamily of homing endonucleases, which cleave DNA site specifically and initiate a process of transferring a mobile intervening sequence into a homologous allele that lacks the sequence. The H-N-H motif contains two anti-parallel b-strands linked to a C-terminal a-helix, with a divalent metal ion located in the center. It is however not clear how the H-N-H motif mediates its function in DNA binding and hydrolysis. For a better understanding of the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 in complex with its inhibitor Im7 (nuclease-ColE7/Im7) in two different crystal forms and we resolved the structures of EDTA-treated, Zn2+-bound and Mn2+-bound complexes in the presence of phosphate ions at resolutions of 2.6 to 2.0. Å. The structure study of the EDTA-treated nuclease-ColE7/Im7 offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. We showed that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg2+-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in H-N-H motif. However a Zn2+ or Mn2+ ions were observed in the center of the H-N-H motif in cases of Zn2+ or Mn2+-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis. We also construct a structural model for the nuclease-ColE7 bound to DNA and propose a mechanism for DNA hydrolysis catatalyzed by the H-N-H endonucleases.
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Nogueira, Gonçalo Raimundo. "Study of the self-assembly of the pro-inflammatory S100A9 protein driven by metal ion binding." Master's thesis, 2016. http://hdl.handle.net/10451/25177.
Full textAbstract:
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016A causa de doenças neurodegenerativas está muitas vezes associada à formação de agregados proteicos e estruturas amilóides. Um exemplo mais representativo desta situação é a doença de Alzheimer, que se caracteriza pela existência de um cenário de neuro-inflamação e amiloidogénese. Neste contexto biológico, ocorre a acumulação do péptido β-amilóide (Aβ) em placas extracelulares e a deposição da proteína tau na forma híper-fosforilada em emaranhados neurofibrilares intracelulares. Como consequência deste fenómeno, o principal sintoma da doença é a deterioração das capacidades cognitivas, porém, os mecanismos subjacentes a estes sintomas não são ainda totalmente compreendidos. Além disso, a desregulação da homeostase dos metais é também observada em pacientes que sofrem desta patologia. A proteína S100A9 tem vindo a ser frequentemente associada com a doença de Alzheimer devido ao seu papel tanto como agente pro-inflamatório e amiloidogénico. A S100A9, também conhecida como Mrp14, pertence à grande família das proteínas S100, as quais possuem dois domínios EF-hand de ligação a cálcio (Ca2+), ligando também zinco (Zn2+) e cobre (Cu2+) em locais distintos dos locais de ligação do Ca2+. Esta proteína é uma das mais potentes proteínas pro-inflamatórias da família S100, sendo sobrexpressa em cenários inflamatórios, incluindo a inflamação decorrente na doença de Alzheimer. Estudos prévios demonstraram que a proteína S100A9 tem a capacidade de se reorganizar-se (self-assembly) em dímero, tetrâmero e também em estruturas oligoméricas maiores, onde se inclui a formação de fibras amilóides, como resultado da sua amiloidogenicidade inerente. Deste modo, sabendo que esta proteína tem a capacidade de ligar Ca2+ e Zn2+, e que, por sua vez, a ligação dos metais a proteínas é um fenómeno que induz alterações conformacionais na estrutura das mesmas, foi proposto que a interacção entre a proteína S100A9 e os iões metálicos pode ser a causa da agregação amilóide e da citotoxidade que advém deste fenómeno. Assim, neste estudo são dadas evidências de como os metais influenciam a reacção de self-assembly da proteína S100A9, através do uso de técnicas complementares, nomeadamente espectroscopia de fluorescência recorrendo ao uso de diferentes sondas conformacionais (ThT, ANS, LCOs: p-FTAA e h-FTAA), ensaios de turbidimetria, uso de anticorpos conformacionais (OC e A11), análise por cromatografia de exclusão molecular e também microscopia de força atómica (AFM). Numa primeira parte deste estudo, foi necessário expressar e purificar o homodímero da proteína S100A9 de modo a serem obtidas quantidades significantes de proteína pura, para que os subsequentes ensaios pudessem ser efectuados. Para tal, a proteína recombinante humana foi expressa em E.coli, seguindo-se uma sequência de etapas, que permitiram isolar os corpos de inclusão e extrair as proteínas contidas nestes, incluindo a proteína em estudo. O extracto proteico foi após submetido a uma serie cromatografias: cromatografia de dessalinização, para remover o cloreto de guanidina (agente desnaturante), cromatografia de exclusão molecular e cromatografia de troca-iónica. Finalmente, e dado que o principal objectivo deste estudo era avaliar o efeito dos iões metálicos (Ca2+ e Zn2+) na proteína S100A9, foi necessário desmetalar a proteína pura obtida. Para tal, o extracto foi incubado com DTT e EDTA, seguindo-se nova cromatografia de exclusão molecular, de modo a obter a forma pura final da proteína desmetalada. Neste estudo, demonstramos que o Zn2+ tem a capacidade de induzir a agregação da proteína S100A9, quando presente em concentrações superiores à capacidade da sua ligação à proteína S100A9 e quando esta última está presente numa concentração superior a 10μM (concentração critica para agregar). Os agregados formados apresentaram reactividade para com a sonda ThT (usada para detetar estruturas amilóides) e foram visíveis, macroscopicamente, sob a forma de um precipitado branco, conferindo um aspecto turvo, que possibilitou seguimento deste fenómeno por ensaios onde monitorizou a turbidez da solução a 360 nm. A formação deste precipitado ocorreu numa escala de tempo de minutos. Além disso, observou-se que o aumento da quantidade de Zn2+ se correlacionou com um potenciamento do processo de agregação, onde se observou um aumento do sinal em ambos os ensaios (fluorescência e turbidez) e diminuição da fase de inicial (lag phase). Os resultados com as outras sondas conformacionais mostraram estar de acordo com a existência desta agregação. No entanto, apesar de os agregados formados levarem à formação de espécies reactivas à ThT e aos LCOs, os resultados obtidos através de ensaios de seeding, análise por AFM e detecção com os anticorpos conformacionais, OC e A11, sugerem que os agregados formados não são de origem amilóide, mas por outro lado que parecem ser maioritariamente de natureza amorfa. A divergência de resultados pode ser devida ao facto da sonda ThT ter já ter mostrado capacidade de se ligar não só a estruturas amilóides mas também de outra natureza. A possibilidade de estes agregados serem induzidos por interações electrostáticas que afectam a solubilidade da proteína foi excluída, uma vez que as cinéticas de S100A9 em presença de excesso de NaCl não conseguiram reproduzir o mesmo efeito que o Zn2+. Por último, a análise das amostras de S100A9 incubadas com Zn2+ (razão 4:1), por cromatografia de exclusão molecular, foi possível observar a presença de espécies com um tamanho semelhante ao da proteína S100A9 na sua forma tetramérica, o que está concordante com o ensaio de AFM. Estes resultados sugerem que o Zn2+ induz a formação de agregados não amilóides, que precipitam, com um possível papel na quelação do Zn2+. Relativamente ao efeito do Ca2+, sendo este um ligando natural da proteína S100A9, seria expectável que a ligação deste ião metálico não induzisse a agregação amilóide da proteína. De facto, os ensaios de cinética de ligação das várias sondas conformacionais e de imunodetecção pelos anticorpos OC e A11, excluíram a existência de formação de estruturas amilóides. Em concordância, as imagens obtidas por AFM indicam a ocorrência de uma reacção de polimerização do tipo não-amilóide, onde a proteína S100A9 adquire uma aparência semelhante longas “cordas”. Curiosamente, um ensaio controlo usando a forma apo da proteína S100A9, revelou a formação de estruturas semelhantes, mesmo sem a adição de iões metálicos. Estas evidências sugerem uma possível função biológica destes agregados. Curiosamente, quando combinada a ligação de Zn2+ e Ca2+ à S100A9 observou-se um efeito aditivo que se refletiu numa agregação heterogénea, com presença de fibras amilóides e outros agregados intermediários passiveis de serem observados por AFM e de serem detectados pelos anticorpos conformacionais OC e A11. Além disso, todas as sondas mostram reactividade para com a S100A9, mas de uma forma sequenciada, demostrando a complexidade deste fenómeno. Neste caso, à semelhança da agregação induzida apenas pelo Zn2+, a solução tornou-se túrbida com formação de precipitado branco. Por fim, é de salientar que foram efectuados ensaios com EDTA, um agente quelante, para remover os iões metálicos ligados à S100A9 com o objectivo de verificar a dependência dos agregados formados. Assim, foi observada a reversão da reacção de self-assembly pelo desaparecimento da turbidez, diminuição da fluorescência da ThT e diminuição dos oligómeros/estruturas observadas por AFM. Em suma, este estudo contribuiu para revelar possíveis mecanismos, pelos quais a ligação de Zn2+ e/ou Ca2+ à proteína S100A9 influencia a sua reacção de self-assembly. Abrindo, assim, caminho para investigação dos papéis dos vários agregados em condições patológicas, nomeadamente na doença de Alzheimer, e para evidenciar o papel e a relevância da proteína S100A9 no despoletar da doença.
S100A9 is a Ca2+ and Zn2+-binding protein which has been increasingly associated with Alzheimer’s disease due to its dual roles as a pro-inflammatory and amyloidogenic agent. This neurodegenerative condition is characterized by neuroinflammation, amyloidogenesis and also disturbance of metal homeostasis. Previous studies have shown that S100A9 is capable to undergo self-assembly into dimer, tetramer and larger oligomers, including formation of amyloid fibrils as a result of its inherent amyloidogenicity. Interestingly, the formation of these different conformational states is thought to be regulated by Ca2+ and Zn2+ binding. Herein, we provide insights of how binding of these metal ions influences S100A9 self-assembly reactions using a set of complementary techniques, including fluorescence spectroscopy with different conformational dyes, conformational antibodies, SEC analysis, turbidimetry assays, and AFM imaging. The results obtained suggest that Zn2+ binding induces the formation of S100A9 assemblies and precipitates; albeit these exhibited ThT-reactivity, AFM imaging elicited mostly amorphous aggregates rather than amyloidogenic fibril structures. SEC analysis of the formed oligomers indicated a size corresponding to that of the S100A9 tetramer, a finding corroborated by AFM measurements. Regarding Ca2+-binding effects, thioflavin-T (ThT)-binding kinetics indicate the occurrence if a polymerization reaction, which leads to the formations of string-like structures as noted by AFM. Interestingly, in control experiments using apo-S100A9, we observed that these string-like structures are also formed upon reaction in the same conditions with no added metal ions. When exposed to both Zn2+ and Ca2+, we noted that S100A9 forms heterogeneous self-assemblies, as inferred from reactivity with different fluorophores including luminescent conjugated oligothiophene dyes which are able to detect a wide range of amyloidogenic protein aggregates. Interestingly, we observed that metal ion chelation using EDTA fully reverts the self-assembly reaction, as shown by disappearance of turbidity, decrease in ThT emission and a decrease on AFM-observable oligomers/structures. Altogether, the results from this work contribute to unveil possible mechanisms through which Zn2+ and Ca2+ binding influences S100A9 self-assembly reaction and will open new avenues for investigations on the roles of such assemblies in pathophysiological conditions.
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Kannappan, Ramakrishnan. "Design and analysis of an electronically switchable ion exchange system." 2009. http://hdl.handle.net/2152/7834.
Full textAbstract:
Metal contamination is a considerable environmental problem because metals are persistent contaminants. Ion exchange is one of the most commonly used treatment options for trace metal removal. This research develops and evaluates a redox active modified ion exchange system that has the potential to reduce the ionic strength of ion exchange regeneration streams. Poly-L-cysteine (PLC) was selected as the redox active, adsorbing functional group on the surface of a reticulated vitreous carbon (RVC) electrode. PLC is an excellent soft acid metal chelator and is unique in that its thiol groups can form disulfide bonds with each other. The reduction of available thiols changes the metal binding capacity of the peptide since the thiol is the primary binding group. RVC provides a macroporous conductive monolithic resin to support the peptide.
An experimental apparatus was designed to study the properties of this system and estimate performance. Distinct oxidized and reduced states of PLC on the surface of the RVC were confirmed by changes in metal binding characteristics. Adsorption edges showed a sharper pH dependence for the reduced electrode compared to the oxidized electrode from pH 3-7. Adsorption isotherms performed at pH 7 showed increased capacity for the reduced electrode. The change was reversible by chemical and electrical reduction. This difference was confirmed at the molecular level with Cd- EXAFS of oxidized and reduced electrodes. A greater degree of cadmium-sulfur coordination was observed on the reduced electrode and a greater cadmium-oxygen coordination was apparant on an oxidized electrode. A multidentate adsorption model was developed to model the pH dependent behavior of cadmium adsorption on the PLC-RVC surface. Nickel adsorption showed increased adsorption in the oxidized state. The most likely explanation is increased carboxylate complexation. The electronically switchable ion exchange system (ESIE) provides a framework for modifying traditional ion exchange processes. The system has 5 to 10 times less specifc capacity than current ion exchange systems, but uses solutions 10-100 times lower in ionic strength for regeneration. Further studies on the effect of ionic strength on adsorption and current usage are necessary to compare the cost of the ESIE process to traditional ion exchange.text
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Liao, Yi-Hung, and 廖宜鴻. "Understanding the Complexity of Alzheimer’s Disease’s Amyloid-β Fibrillization via Metal Ion Binding, Familial Mutants, and Gold Nanoparticles." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/96790252837984622670.
Full textAbstract:
博士國立臺灣大學
生化科學研究所
102
Amyloid plagues are the major pathogenic hallmarks in many neurodegenerative diseases. Fibrillization of the primary constituents, amyloid-β (Aβ), in plagues is considered the pathogenesis of Alzheimer’s disease (AD). Conclusive evidence is still lacking to reach a comprehensive and convergent view of how Aβ is modulated in the pathogenesis of AD. Here, in three separate chapters, we examined how gold nanoparticles (AuNPs) interfere with fibrillization of Aβ, the effects of single-point N-terminal mutation, H6 and D7, on the interactions between metal ions and Aβ, and how single-point mutations, E22 and D23, influence the structures of fibril. In Chapter 3, experimental results showed that (1) bare AuNPs inhibited Aβ fibrillization in a dose-dependent manner and redirected Aβ forming fragmented fibrils and spherical oligomers; (2) bare AuNPs bound preferentially to Aβ fibrils but not amorphous aggregates; (3) negative surface potential of AuNPs was required for inhibitive effect. In conjunction with our in vitro biophysical and biochemical data, cell studies from our lab member revealed that in our experimental settings, inhibition of Aβ fibrillization by negatively charged AuNPs also reduce Aβ-induced cytotoxicity in neuronal cells. In Chapter 4, we aimed to reveal distinctions among structures of homopolymeric fibrils (homo-fibrils) formed from incubation of monomers of a single species, and heteropolymeric ones (hetero-fibrils) started from incubation of equal-molar-mixed monomers of wild type and an Aβ variant. We showed that E22 hetero-fibrils are distinct from E22 homo-fibrils in terms of secondary structures. Perturbed secondary structure could influence conformational packing of cross β-sheet, which were not revealed by stoichiometry between Aβ and ThT but by ThT fluorescence lifetime. In Chapter 5, we investigated millisecond binding kinetics of four metal ions, Zn2+, Cu2+, Fe3+, and Al3+. We then focused on Cu2+ and demonstrated that mutation at residues H6 and D7 impact on Cu2+ binding affinities. We found by Bis-ANS fluorescence that diminished hydrophobic exposure was accompanied by Cu2+ binding except for D7A and H6R. Our preliminary proton NMR showed that there existed environmental variance surrounding residues H6, H13/H14, and Y10 among monomeric Aβ variants in native condition. Cu2+-binding-induced peak loss was less evident in D7 mutants. 1H-15N NMR HSQC spectroscopy provided additional information regarding the residual involvement on Cu2+ binding between D7H and wild type Aβ.
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Chakraborty, Twarita. "Molecularly Imprinted Polymers Based On Fluorescent And Template Binding Cross-Linker." Thesis, 2007. http://etd.iisc.ernet.in/handle/2005/2006.
Full textAbstract:
The synthesis of materials with molecular recognition properties has become a topic of great technological and scientific interest. Molecular imprinting is one of the most effective strategies in preparing highly selective synthetic receptors. The technique of molecular imprinting involves the copolymerization of functional and cross-linking monomers in the presence of a molecular template. Following polymerization and subsequent removal of the template, the molecularly imprinted polymer (MIP) retains a “molecular memory” of the template. During rebinding, the resultant polymer shows higher affinity and selectivity towards the molecular template when compared to other structural analogs. Ease of preparation and high thermal and chemical stability of this class of materials offers a broad range of potential applications. Promising areas of application include separation, chromatography, catalysis, sensors, antibody mimics, and drug delivery etc.
The thesis entitled “Molecularly Imprinted Polymers based on Fluorescent and Template binding Cross-linker” deals with the design and synthesis of several molecularly imprinted polymers (MIPs) using different functional and cross-linking monomers, the main focus being use of preformed template-monomer complex, use of fluorescent cross-linker and development of functional group containing cross-linker.
Chapter 1: An Introduction to Molecularly Imprinted Polymers.
The first chapter provides an introduction to the field of molecularly imprinted polymers. It presents an overview of molecular imprinting process including a brief history of its discovery and its evolution to the present form. This chapter further elaborates on the principle of molecular imprinting with an emphasis on different parameters that directly affect their performance. It also provides a brief review of the applications of molecularly imprinted polymers.
Chapter 2: Highly Cross-linked Metal Ion Imprinted Polymers.
The second chapter deals with the synthesis of series of highly cross-linked metal-ion imprinted polymers. The process of metal ion-imprinting usually involves carrying out the polymerization and cross-linking directly in presence of the appropriate metal ion. In the present study, chemical-immobilization method was adopted which involves the use of preformed metal complexes with polymerizable group for the imprinting. Acrylate complexes of various metal-ions, such as Cu2+, Zn2+, Co2+, Ni2+, Pb2+ and Cr3+, were synthesized prior to polymerization. These pre-assembled complexes were then used to prepare MIPs, in the anticipation that this would lead to enhanced selectivity. Ethyleneglycol dimethacrylate (EGDMA) was used as the cross-linking monomer. As a control, the respective non-imprinted polymers (NIPs) were also made in absence of the template metal ion. Following polymerization, the template metal ion was extracted from the resultant metal ion-imprinted polymer. The selectivity of the metal ion-imprinted polymers was examined by a batch process using analytical tools, such as, Atomic Absorption Spectroscopy (AAS) and Inductively Coupled Plasma Spectroscopy (ICP). The spectroscopic studies revealed significant selectivity of all the MIPs towards the template metal ion. Among all six metal ion-imprinted polymers, Pb2+ and Cr3+ ion-imprinted polymer showed remarkable selectivity, followed by Cu2+ and Zn2+ ion-imprinted polymers. The Co2+ and Ni2+ ion-imprinted polymers exhibited comparatively poor selectivity. Representative plots depicting the selectivity exhibited by Pb2+ and Cr3+ ion-imprinted polymers are shown in Figure 1. These observations were rationalized based on the size and geometric preferences imposed by the imprinted site on the ion that binds to it.
Figure 1. Selectivity study for (a) Pb2+ ion-imprinted polymer, (b) Cr3+ ion-imprinted polymer.
Chapter 3. Molecularly Imprinted Fluorescent Chemosensor for Copper (II).
Cu(II) is a source of important pollutant and therefore, the development of sensors that can detect Cu(II) selectively as well as remove Cu(II) from contaminated samples is an important objective. The use of molecular imprinting technique is an appealing approach in this regard. For this, a fluorophore containing cross-linker, namely 9,10-bis-(acryloyloxymethyl)anthracene (BAMA) was synthesized. This fluorescent cross-linker was used along with the standard cross-linker, EGDMA, for preparing Cu2+ ion-imprinted polymer. The complex of copper methacrylate (Cu-MAA) was prepared prior to polymerization used for the preparation of MIP. The resultant imprinted polymer exhibited quenching of the fluorescence in presence of Cu2+ ion, both in organic and aqueous medium. The efficiency of quenching of NIP (prepared in absence of Cu2+ ion) was significantly lower than that of MIP. A typical stack spectra showing the quenching process, along with a comparison of the quenching efficiency of MIP and NIP is shown in Figure 2.
The imprinted polymers showed significant selectivity over other non-template metal ions, thereby reaffirming the importance of the imprinting process. The sensitivity of the fluorescence detection could be enhanced by increasing the level of the fluorophore incorporation. The increased sensitivity in detecting Cu2+ ion, demonstrated by the MIP suggests that a statistically random incorporation of the fluorophore into MIP matrices could be a useful approach for imparting a sensing element to MIPs.
Figure 2. Fluorescence spectra of the (a) imprinted (MIP-1) and (b) non-imprinted (NIP-1) polymers in the presence of various concentration of Cu(OAc)2 in methanol. (c) Comparison of quenching efficiency of MIP-1 and NIP-1. Data were collected 3 h after addition of copper solution. I0 and I are the fluorescence intensities at 399 nm of the polymers in the absence presence of copper respectively. Two individual runs are presented in (c).
Chapter 4. Molecularly Imprinted Turn-Off-On Sensor.
This chapter describes the design and synthesis of molecularly imprinted fluorescent turn-off-on sensor utilizing the same fluorescent cross-linker, BAMA. Combining the process of fluorescence resonance energy transfer (FRET) with molecular imprinting technique, a novel turn-off-on sensor was developed. A molecularly imprinted polymer was prepared using a fluorescent template Coumarin-30 (C-30). C-30 was chosen as the template to ensure a significant overlap of the emission spectra of BAMA and the absorption spectra of C-30, thereby optimizing for FRET.
Figure 3. Structures of relevant molecules.
The C-30 imprinted polymer exhibited simultaneous quenching in fluorescence (turn-off) of BAMA and enhancement in fluorescence (turn-on) of C-30 (Figure 4). The imprinted polymer showed significantly better performance over the non-imprinted polymer (NIP).
Figure 4. Fluorescence spectra of the (a) imprinted (MIP) and (b) non-imprinted (NIP) polymers with increasing concentration of the template Coumarine-30 in methanol.
The UV-vis studies revealed that the more effective quenching is indeed due to the affinity for C-30 exhibited by the higher binding imprinted polymer. The imprinted polymer also showed significant selectivity over structurally analogous molecules. Therefore, both high sensitivity and selectivity were realized in such novel off-on sensor. Extension of this concept to other biologically relevant fluorescent templates could lead to potentially useful applications.
Chapter 5. Design of New Template Binding Cross-linker.
In molecularly imprinted polymers (MIP), high cross-linking density (~80 to 90 mole percent) is essential to ensure high selectivity, which limits the functional (binding) monomer to about 10-20 mole percent. Methacrylic acid (MAA) and ethyleneglycol dimethacrylate (EGDMA) are the most common combination of functional monomer and cross-linker, respectively, used in molecular imprinting. Generally a molecularly imprinted polymer made with this combination, contains only 10-20% binding sites. This limitation of binding site density is an aspect that has largely been overlooked. In order to improve the efficiency of MIP materials by enhancing the number of binding sites, a new cross-linking monomer (CYDI, 1) with two carboxylic acid groups was designed and synthesized by coupling itaconic anhydride with cyclohexane dimethanol (Figure 5).
Figure 5. Structures of relevant molecules. The new functional group bearing cross-linking monomer (1) Itaconate ester of cyclohexanedimethanol (CYDI), the template (2) theophylline (Theop) and the structural analogue of template (3) caffeine (Caff).
This new cross-linking monomer was then employed for preparing molecularly imprinted polymer using a drug molecule, theophylline (Theop 2, a bronchodilator) as the template. Seven molecularly imprinted polymers were synthesized with different ratios of CYDI and EGDMA, keeping the cross-linking density constant. The binding efficiency and the selectivity of these imprinted polymers were thoroughly investigated. It was seen that while saturation binding values for theophylline increased continuously with functional cross-linker (CYDI) content, the optimum selectivity with respect to analogous substrate, caffeine, was attained at 40 mol% CYDI. These studies suggest that the approach of using functional group containing cross-linkers could lead to improved MIP performance.
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Casciato, Shelly Lynn 1984. "Development of a fluorescence model for the determination of constants associated with binding, quenching, and FRET efficiency and development of an immobilized FRET-peptide sensor for metal ion detection." Thesis, 2012. http://hdl.handle.net/2152/ETD-UT-2012-08-6087.
Full textAbstract:
This thesis presents a modeling program to obtain equilibrium information for a fluorescent system. Determining accurate dissociation constants for equilibrium processes involving a fluorescent mechanism can prove to be quite challenging. Typically, titration curves and non-linear least squares fitting of the data using computer programs are employed to obtain such constants. However, these approaches only consider the total fluorescence signal and often ignore other energy transfer processes within the system. The current model considers the impact on fluorescence from equilibrium binding (viz., metal-ligand, ligand-substrate, etc.), quenching and resonance energy transfer. This model should provide more accurate binding constants as well as insights into other photonic processes. The equations developed for this model are discussed and are fit to experimental data from titrimetric experiments. Since the experimental data are generally in excess of the number of parameters that are needed to define the system, fitting is operated in an overdetermined mode and employs error minimization (either absolute or relative) to define goodness of fit. Examples of how changes in certain parameters affect the shape of the titrimetric curve are also presented. The detection of metal ions is very important, causing a need for the development of a metal ion sensor that provides selectivity, sensitivity, real-time in situ monitoring, and a flexible design. In order to be able to perform in situ monitoring of trace metal ions, FRET-pair labeled peptides were attached to a Tentagel[trademark] resin surface. After soaking in nonmetal and metal solutions (pH = 7.5), the resin beads gave an enhanced response in the presence of Hg²⁺ and Zn²⁺. Using a t-test, the signals of the beads that were soaked in a solution of each of these metal ions (and that of Cd²⁺) were determined to be significantly different from beads soaked in a solution without metal. However, the standard deviation between a set the beads was too large in order to differentiate a bead that was soaked in nonmetal solution versus one soaked in a metal containing solution.text
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Zhang, Mingxuan. "Characterization of structural changes and large-scale protein dynamics and their influence on metal ion binding by human serum transferrin by ESI MS." 2007. https://scholarworks.umass.edu/dissertations/AAI3254945.
Full textAbstract:
Human serum Transferrin (hTf) is a ∼80 kDa protein, whose function is iron sequestration and transport. The two lobes of the protein (commonly referred to as N- and C-lobes) have a very high degree of structural identity and provide two distinct binding sites for a ferric ion. In addition to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study ESI MS is used to investigate interactions between human serum transferrin and two non-ferrous metals (indium and bismuth), conformational changes upon metal binding, as well as characterize human serum transferrin N-lobe (hTf/2N) global dynamics and functionally important local dynamic events. The In-hTf complex was directly detected by ESI MS; the Bi-hTf complex in solution was established by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in solution. The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination which causes anomalous instability of the transferrin-bismuth complex in the gas phase. The apo-hTf/2N and Fe-hTf/2N were used in hydrogen/deuterium exchange (HDX) measurement for characterizing protein dynamics. In this measurement, back-exchange was corrected for every transferrin N-lobe peptic fragment individually. The results showed that iron binding induce more compact conformation and significant decrease of HDX kinetics around hinge regions and Lys206 which is one amino acid from the dilysine-trigger. However, the changes around iron binding sites are not as significant. Our hypothesis is that instead of having frozen states, transferrin has certain frequency of conformational hopping. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures was developed utilizing the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode.
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"Nanopore Sensing Of Peptides And Proteins." Thesis, 2013. http://hdl.handle.net/10388/ETD-2013-11-1280.
Full textAbstract:
In recent years the application of single-molecule techniques to probe biomolecules and intermolecular interactions at single-molecule resolution has expanded rapidly. Here, I investigate a series of peptides and proteins in an attempt to gain a better understanding of nanopore sensing as a single-molecule technique.
The analysis of retro, inversed, and retro-inversed isomers of glucagon and α-helical Fmoc-D2A10K2 peptide showed that nanopore sensing utilizing a wild-type α-hemolysin pore can distinguish between all four isomers while circular dichroism can only distinguish between chiral isomers, but not between directional isomers.
The investigation of a series of proteins of different chemical and physical properties revealed important information about nanopore analysis of proteins. Contrary to some reports in the literature, all proteins analysed here induced large blockade events. The frequency of total events and the proportion of large blockade events were significantly reduced in tris(hydroxymethyl)aminomethane or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffers and were only restored by the addition of ethylenediaminetetraacetic acid or the use of phosphate buffer, both of which can sequester metal ions. Furthermore, the results obtained with the proteins in the presence of ligands demonstrated that transient or partial unfolding of proteins can be detected by nanopore analysis confirming the usefulness of this technique for conformational studies or for protein/ligand interactions. Interestingly, while the blockade current histograms were different for each protein there was no obvious correlation between the properties of the proteins and the blockade current histograms.
In an attempt to identify whether the large blockade events were translocation or intercalation, both an indirect and a direct approach were taken. The indirect approach which relies on the effect of voltage on the interaction of the molecule with the pore provided no conclusive answer to the question of protein translocation through the α-hemolysin pore. In contrast, the direct approach in which ribonuclease A is added to the cis side of the pore and then the trans side is tested for enzyme activity showed that ribonuclease A doesn't translocate through the α-hemolysin pore.