Spelling suggestions: "subject:"carbonic anhydride II""
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The interaction of human carbonic anhydrase II to solid surfaces and its applicationsUdd, Annika January 2009 (has links)
<p><p>The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO<sub>2</sub>-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.</p></p>
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The interaction of human carbonic anhydrase II to solid surfaces and its applicationsUdd, Annika January 2009 (has links)
The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO2-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.
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Bone Phenotype of Carbonic Anhydrase II Deficient and Calbindin-D28k Knockout Mice and Development of a Method to Measure In Vivo Bone Strains in MiceMargolis, David Stephen January 2008 (has links)
Since the development of knockout and transgenic mouse models, mice have become the most widely used mammalian animal model to study bone. Despite the advances in knowledge of bone biology and function that have occurred from use of mouse models, many studies use primarily qualitative techniques, which may result in overlooking important subtle pathophysiologic changes. The hypothesis of this dissertation is that quantitative techniques to measure bone structure and function could identify the physiologic role of carbonic anhydrase II and calbindin-D28k in mouse bone, despite earlier qualitative studies indicating mice without these proteins have normal bone structure and function. Furthermore, a method to quantify bone function in vivo will be tested in a mouse model.Although carbonic anhydrase II deficient mice are less severely affected than patients, the mice demonstrate features of osteopetrosis including metaphyseal widening and a 50% increase in trabecular bone volume. The mice partially compensate for inhibited osteoclast function by increasing osteoclast number.Calbindin-D28k knockout mice demonstrated an increase in bone volume that results from additional bone at the endosteal surfaces. The higher bone volume results in increased stiffness and failure loads, highlighting the potential use of drugs that inhibit calbindin-D28k to treat diseases such as osteoporosis.Finally, calcium phosphate ceramic and hydroxyapatite particles used as strain gauge coatings demonstrated bone bonding to mouse femora after two months in vivo. The use of hydroxyapatite particles to coat strain gauges is the first time this method has been used with all commercially available materials, and will allow other research groups to use this technique. The major limitation to in vivo bone strain measurement in mice is the relatively large size of the sensors, which resulted in increased second moments of inertia in the implanted bones.Overall, this dissertation demonstrates that the use of quantitative techniques, including histology, histomorphometry, µCT imaging, and mechanical testing can measure subtle changes in bone properties that have been previously overlooked. Development of additional quantitative methods to study bone biomechanics in mouse models may encourage other research groups to quantify bone properties if no changes are noted using primarily qualitative methods.
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Human Carbonic Anhydrase Ii; Preparation, Metal-Substitution, Activity, and InhibitionWilson, David L 14 August 2015 (has links)
This report details the activities and inhibition of metal-substituted human carbonic anhydrase II (M-HCA-II). The traditional activities (hydrolysis of CO2 and para-nitrophenol acetate) in addition to new activities (oxidation of 2-aminophenol, disproportionation of H2O2, and disproportionation of superoxide) were investigated. Values reported for the relative hydrolytic activities of M-HCA-IIs are reported here for the first time, ranging from 47.5 % (plus or minus 0.6) to 86 % (plus or minus 4) for the hydrolysis of CO2 and from 0.299 % (plus or minus 0.012) to 4.72 % (plus or minus 0.015) for the hydrolysis of para-nitrophenol acetate. With respect to new activities, only the oxidation of 2-aminophenol was observed. Turnover was observed for Fe-HCA-II (kcat/KM = 3.6 plus or minus 1.3 mM-1 s-1) and Cu-HCA-II (kcat/KM = 8 plus or minus 2 mM-1 s-1). Inhibition of Zn-, (di-substituted) Cu2-, and Cu/Zn-HCA-II hydrolysis of CO2 and para-nitrophenol acetate by sulfanilamide, coumarin, and ortho-coumaric acid were investigated. Sulfanilamide was shown to inhibit: Zn-HCA-II, Cu2-HCA-II, and Cu/Zn-HCA-II - (with CO2) KM = 8.9 plus or minus 1.1 microM, 11 plus or minus 2 microM, 8.8 plus or minus 1.4 microM and (with p-nitrophenyl acetate) KM = 8.4 plus or minus 1.0 microM, (none), 8.4 plus or minus 1.4 microM, respectively. No inhibition was observed for coumarin or ortho-coumaric acid or its derivatives for any CAs studied.
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Reconnaissance de surfaces protéiques par des foldamères d'oligoamides aromatiques / Recognition of protein surfaces by aromatic oligoamide foldamersBuratto, Jeremie 07 January 2014 (has links)
Les interactions protéine - protéine sont au centre de nombreux processus biologiques, et représentent des cibles thérapeutiques pertinentes pour le traitement de certaines maladies. La conception de molécules antagonistes visant à inhiber ces interactions requiert la reconnaissance spécifique d’une des surfaces protéiques impliquées. Les foldamères de type oligoamides de quinoline constituent de bons candidats. Leur production et leur fonctionnalisation sont relativement aisées. Ils adoptent des structures hélicoïdales semblables à celles rencontrées dans les protéines. Grâce à différentes techniques biophysiques (CD, SPR, diffraction des rayons X), nous montrons que ces molécules sont aptes à reconnaître une surface protéique. Deux protéines cibles ont été choisies : l’interleukine 4 et l’anhydrase carbonique humaine II.La stratégie retenue dans ce travail (ancrage du foldamère à la protéine via un bras espaceur) nous a permis d’obtenir des informations structurales sur les interactions foldamère – protéine avant toute optimisation de ces interactions. La première structure 3D d’un complexe foldamère de quinoline complexée à une protéine est décrite. / Protein-protein interactions are a central issue in biological processes and represent relevant therapeutic targets for the treatment of diseases. The design of antagonistic molecules directed towards the disruption of these interactions requires the specific recognition of protein surfaces. Quinoline oligoamide foldamers present all the properties to reach that point. They are easily synthesized and fold into helices (similar to α helices) which can be decorated. Thanks to biophysical studies (CD, SPR, RX diffraction), we demonstrate that these molecules are able to recognize protein surfaces. Two proteins have been studied: the human interleukin 4 and the human carbonic anhydrase II.The applied strategy (keeping the foldamer close to the protein surface via a linker) allowed us to gather structural information about foldamer protein interactions before any strong binding is established. The first crystal structure between a protein and an aromatic amide foldamer is reported.
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Development of a label-free biosensor method for the identification of sticky compounds which disturb GPCR-assaysMohammed Kader, Hamno January 2013 (has links)
It is widely known that early estimates about the binding properties of drug candidates are important in the drug discovery process. Surface plasmon resonance (SPR) biosensors have become a standard tool for characterizing interactions between a great variety of biomolecules and it offers a unique opportunity to study binding activity. The aim of this project was to develop a SPR based assay for pre-screening of low molecular weight (LMW) drug compounds, to enable filtering away disturbing compounds when interacting with drugs. The interaction between 47 LMW compounds and biological ligands were investigated using the instrument BiacoreTM, which is based on SPR-technology.
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Bioanalytical Applications of Intramolecular H-Complexes of Near Infrared Bis(Heptamethine Cyanine) DyesKim, Junseok 15 July 2008 (has links)
This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.
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Reconnaissance de surfaces de protéines par les foldamères d'oligoamides aromatiques / Protein surface recognition using aromatic oligoamide foldamersVallade, Maëlle 29 September 2016 (has links)
Les protéines étant au coeur d’un grand nombre de processus biologiques, elles sont des cibles thérapeutiques largement convoitées. Les foldamères, notamment les oligoamides aromatiques, présentent une structure bien définie, prévisible, stable en solution et à l’état solide. Ajouté à cela, leur taille moyenne en fait de bons candidats pour la reconnaissance de surfaces de protéines, grâce à leurs chaînes latérales protéinogènes. Cette thèse présente les différentes étapes de leur conception, de la synthèse de la brique constitutive à l’obtention d’un foldamère fonctionnalisé grâce à la synthèse en phase supportée. La stratégie d’investigation des interactions entre un foldamère et une protéine est détaillée. L’originalité réside dans le fait que le foldamère est ancré directement à la protéine et le dichroïsme circulaire sert de méthode de screening. L’analyse structurale des hits permet de générer de nouveaux foldamères dans le but d’améliorer les interactions avec la protéine : c’est une stratégie itérative. Cette approche est appliquée premièrement à l’anhydrase carbonique humaine II, protéine modèle qui sert de preuve de principe pour cette approche ; puis à des protéines d’intérêt thérapeutique plus important : l’interleukine 4 et la cyclophiline A. Enfin, une étude concernant l’introduction de flexibilité au sein de foldamères de quinolines est présentée. / Since proteins are at the basis of many biological processes, they are widely studied as therapeutic targets. Aromatic oligoamide foldamers have a very well defined structure, predictable and stable both in solution and solid state. Because of their medium size, they appear as potent candidates for protein surface recognition thanks to their proteinogenic side chains. This manuscript presents the different steps of their design, from the scaffold’s synthesis to obtaining a functionalized foldamer, thanks to solid phase synthesis. The strategy to investigate protein/foldamer interactions will be detailed. Its originality lies in the fact that the foldamer is anchored to the protein. Circular dichroism has been used as a screening method to detect foldamer/protein interactions. Structural analysis of the hits will allow the design of new foldamers with the objective of enhancing foldamer/protein interactions: it is an iterative strategy. This approach has been applied firstly to human carbonic anhydrase II (HCA). This protein is used as a model system and proof of concept before moving to more therapeutically relevant proteins; interleukin 4 and cyclophilin A. Finally, a study on introducing flexibility in quinoline foldamers is presented.
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