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Molecular Mechanism Of Incorporation Of Factor Va Into Prothrombinase, Melissa Barhoover

ETD Archive

The mainstay of the blood coagulation cascade is the formation of the fibrin clot, catalyzed by the serine protease, thrombin. The prothrombinase complex catalyzes the activation of prothrombin to thrombin and, is composed of the enzyme, factor Xa, and the protein cofactor, factor Va, in the presence of divalent metal ions associated on a membrane surface. The enzyme, factor Xa, alone can activate prothrombin by two sequential proteolytic cleavages at Arg271 and Arg320 resulting in the intermediates, Fragment 1.2 and Prethrombin 2. The overall rate of this reaction is not compatible with survival. On the other hand, the incorporation of …

Characterization Of A Type Ii Metallothionein From Helianthus Annuus Using Recombinant Dna Techniques, Sridhar Bhogavalli

ETD Archive

Metallothioneins (MTs) are ubiquitous low molecular weight, cysteine rich proteins with a pronounced affinity for metal ions with d10 configuration such as Cu, Cd and Zn. These heavy metals form metal-thiolate clusters with cysteine side chains. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. We describe here a method of isolation and purification of metallothionein from the plant Helianthus annuus, performed by affinity chromatography using glutathione-agarose column. In this study, alignment of gene sequence of the isolated H. annuus metallothionein cDNA with known MTs showed that it belongs …

Characterizatin Of A Type Ii Metallothionein From Helianthus Annuus Using Recombinant Dna Techniques, Sridhar Bhogavalli

ETD Archive

Metallothioneins (MTs) are ubiquitous low molecular weight, cysteine rich proteins with a pronounced affinity for metal ions with d10 configuration such as Cu, Cd and Zn. These heavy metals form metal-thiolate clusters with cysteine side chains. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. We describe here a method of isolation and purification of metallothionein from the plant Helianthus annuus, performed by affinity chromatography using glutathione-agarose column. In this study, alignment of gene sequence of the isolated H. annuus metallothionein cDNA with known MTs showed that it belongs …

Protein Free Energy Landscapes Remodeled By Ligand Binding, Troy C. Messina, David Talaga

Department of Chemistry and Biochemistry Faculty Scholarship and Creative Works

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three components at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization, and literature work were used to assign the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via …

Synthesis Towards Novel Boron Modified Polypeptides As Potential Hiv-1 Protease Inhibitors, Tajinder Heer

Undergraduate Honors Theses

Although current HIV-1 protease inhibitors on the market show high specificity, a number of side effects can result from their administration. Furthermore, quick drug resistance is developed due to mutations of the protease. Consequently, there exists a great and urgent need for the development of a novel HIV-1 protease inhibitors that have less toxicity, greater bioavailability, and exhibit distinct resistance profiles. Recent studies have shown that borinic acid HIV-1 protease inhibitors, which can act both associatively and competitively, have a higher affinity for HIV-1 protease variants than compounds that are strictly transition state analogs. Furthermore, such inhibitors were found to …

Synthesis Of Nitrogen-Containing Heterocycles From The Reaction Of Amidrazones With \Alpha-Haloesters, Hany M. Dalloul, Peter H. Boyle

Turkish Journal of Chemistry

The reaction of amidrazones 1 with \alpha-haloesters 2 formed 1,3,5-substituted 4,5-dihydro-1,2,4-triazin-6-ones (4a-z) rather than 1,2,4-triazin-5-one derivatives (5a-z). The microanalysis and spectral data of the synthesized compounds are in full agreement with their molecular structure.

Synthesis And Cytotoxic Activity Of Platinum(Ii) And Platinum(Iv) Complexes With 2-Hydroxymethylbenzimidazole Or 5(6)-Chloro-2-Hydroxymethylbenzimidazole Ligands Against Mcf-7 And Hela Cell Lines, Semra Utku, Fatma Gümüş, Si̇bel Gür, Aykut Özkul

Turkish Journal of Chemistry

Four platinum(II) and 4 platinum(IV) complexes with the structures [PtL_2Cl_2], [PtL_2I_2], [PtL_2Cl_4], and [PtL_2Cl_2(OH)_2] (L = 5(6)-non/orchloro-substituted-2-hydroxymethylbenzimidazole ligands as ``non-leaving groups''), respectively, were synthesized and characterized by their elemental analyses, and IR and ^1H-NMR spectra. In vitro cytotoxic activities of the platinum(II) and platinum(IV) complexes were tested against the human MCF-7 (breast cancer) and HeLa (cervix cancer) cell lines using the cell culture method. In general, the platinum(II) complexes were more active than the corresponding platinum(IV) complexes. The complexes, which were found to be less active than cisplatin, exhibited cyctotoxicity comparable to carboplatin on the human MCF-7 and HeLa cell …

Sulfite Reduction In Mycobacteria, Rachel Pinto, Joseph S. Harrison, Tsungda Hsu, William R. Jacobs Jr., Thomas S. Leyh

College of the Pacific Faculty Articles

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict …

Nitroreductase: Evidence For A Fluxional Low-Temperature State And Its Possible Role In Enzyme Activity, Peng Zhang

University of Kentucky Doctoral Dissertations

The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as riboflavin derivatives, using a tightly-bound flavin mononucleotide (FMN) cofactor. It has important environmental and clinical implications. Previous studies have focused on elucidating NRs catalytic mechanism and solving its crystal structure.

In this dissertation work, we first develop and implement new strategies for labeling NR with stable isotopes, and report a completely re-designed protocol for NRs purification. Then we report the successful assignment of over half of NRs backbone resonances using 3d-NMR methods. The most significant …

Abstraction Of Dimensional Relevance Of Molecular Representation: Generation Of Novel Inhibitors Against Malarial Plasmodium Falciparum Plasmepsin Iv, Christopher B. Abdul-Wahid

Undergraduate Honors Theses

The malarial parasite Plasmodium falciparum is endemic to 50% of the world's population, killing three million people a year, many of whom are children under five years of age. Due in part to the development of malarial resistance to current drug therapies, novel drug targets are being sought. The malarial hemolytic aspartic protease Plasmepsins are such targets of rational drug design.

We are developing a methodology to rapidly generate combinatorial libraries of chemical structures using a one-dimensional representation (Simplified molecular input line entry specification (SMILES)) of a molecule, to automatically generate a minimized three-dimensional representation (SYBYLMOL2), and to screen this …

Synthesis Of Novel Aziridine Derivatives Of Podocarpic Acid, Stephen Rhoden

Electronic Theses and Dissertations

Podocarpic acid (a diterpenoid resin acid extracted from the Podocarpacea specie of plants) has shown cytotoxicity against carcinoma of the nasopharynx. Since this discovery has been made, research has been performed in order to alter the structure of the resin acid so as to increase the anticancer activity. The carboxylic acid and phenol functional groups, which are present in podocarpic acid, make it possible to synthesize new derivatives selectively at the C-15, C-13, and C-7 positions as well as by substitution of the phenol hydroxyl group. Thus numerous derivatives can be prepared, in high yield, for the purpose of investigating …

Membrane Fragmentation By A 20-29 Fragment Of Human Islet Amyloid Polypeptide And Rat Amyloid Polypeptide, Shyamprasad Samisetti

Master's Theses and Doctoral Dissertations

A key factor in the development of type II diabetes is the loss of insulin-producing pancreatic beta cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP, also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill β-cells by disrupting the cellular membrane. In this study we report membrane fragmentation by hIAPP20-29 and the rat version, rIAPP20-29, using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes.

31P NMR results suggest that membrane fragmentation by hIAPP is related to peptide …

The Effect Of The Reducing Agent Triphenylphosphine On Arachidonic Acid Metabolism By Prostaglandin H2 Synthase, Lalini Ailaboina

Master's Theses and Doctoral Dissertations

Prostaglandin H2 synthase (PGHS) catalyzes the conversion of arachidonic acid to prostaglandin endoperoxide (PGH2), which is a precursor to a variety of prostaglandins that function as hormones. Gas chromatography combined with mass spectrometry is employed to look for the novel products produced by reaction of triphenylphosphine with arachidonic acid prior to and during the PGHS reaction. Triphenylphosphine (TPP) is known to reduce hydro peroxides to alcohols and is used in the present study to ascertain the extent to which hydroperoxides in arachidonic acid preparations and in PGHS contribute to enzyme activity. Incubation of PGHS with arachidonic acid that was treated …

Development Of A Microwave-Assisted Epoxide Aminolysis And Investigation Of The Aza-Cope Rearrangement--Mannich Cyclization For Alkaloid Synthesis, Brendan Roberto D'Souza

Master's Theses and Doctoral Dissertations

β-Amino alcohols are important organic compounds with numerous applications. Commonly β-amino alcohols are prepared by ring opening of epoxides using an amine nucleophile and a catalyst or promoter. Ring opening can take place by the SN1 pathway or the SN2 pathway, but no single catalyst or promoter consistently gives SN2 selectivity. Additionally, long reaction times are often required to obtain adequate yields. We have demonstrated that microwave heating reduces reaction times for the aminolysis of both reactive and unreactive, sterically hindered epoxides. These reactions were accomplished with various amines. In all cases, only one equivalent of amine and epoxide were …

Effect Of Counter Ion Concentration Added With Mixture Of Water And Ethylene Glycol On Krafft Temperature Of Sodium Dodecyl Sulfate, Sateesh Krishna Chand Chundru

Master's Theses and Doctoral Dissertations

Surfactants are amphiphilic organic compounds having both hydrophobic and hydrophilic groups. Surfactants in solution form aggregates known as micelles. The minimum temperature at which surfactants forms micelles is known as Krafft temperature. The Krafft temperature of sodium dodecyl sulfate (SDS) was measured at various counter ion concentrations in two solvent systems, water and 10% ethylene glycol. The Krafft temperature of sodium dodecyl sulfate increased in both water and 10% ethylene glycol with increasing counter ion concentration. The Krafft temperature of sodium dodecyl sulfate in 10% ethylene glycol was higher than the Krafft temperature of sodium dodecyl sulfate in water, at …

Synthesis Of An Isotopically Labelled Analog Of The Antimicrobial Peptide Ll-37, Rajesh Penumatcha

Master's Theses and Doctoral Dissertations

LL-37 is a cationic cathelin-associated broad spectrum antibiotic peptide of human neutrophils. Its mechanism of action is by disruption of the bacterial cell membrane structure. LL-21 is a simplified form of the peptide that contains only the core portion of LL-37, which retains antimicrobial activity and is easier to synthesize. We synthesized an 15N –Val-labeled LL-21 peptide which allows us to perform antimicrobial assays to know the antimicrobial activity of the new peptide LL-21 on various gram positive and gram negative bacteria. We found that our new peptide LL-21 shows good minimum inhibitory concentrations on various gram positive and gram …

Jonas Kamlet (1914-1960) Chemist/Entrepreneur, Dean F. Martin, Barbara B. Martin

Chemistry Faculty Publications

Jonas Kamlet, Ph.D., as a chemist, inventor, and a significant entrepreneur who founded Kamlet Laboratories in New York City that became the model of a successful consulting firm. Among his hundreds of patents and inventions were a tablet that could be used to measure glucose in urine, a treated strip that could detect pregnancy, a fodder for ruminants that used newsprint, and biuret (a self-condensation product of urea) that could be used as a non-protein component of fodder. Following his death in an airplane collision on December 16, 1960, his wife and collaborator (and later a resident of Florida) carried …

Concomitant Polymorphism In 1,2-Bis(9-Anthryl)Acetylene, Rebecca Goldstein

Honors Theses

This thesis details the serendipitous discovery of the phenomenon of concomitant conformational polymorphism found to exist in 1,2-bis(9-anthryl)acerylene. 1,2-bis(9anthryl)acetylene is a previously published molecule. However, the published crystal structure showed the molecule to be completely planar, and to crystallize in the form of orange needles. While this form of the crystal was indeed found during the synthesis of 1,2-bis(9anthryl)acetylene, a new polymorph, crystallizing as yellow prisms, was also discovered. This yellow polymorph was found by X-ray crystallography to be monoclinic with an angle of 66.7 between the anthracene planes. The planar polymorph 1,2-bis(9-anthryl)acetylene was also found by X-ray crystallography to …

Detailed Mechanistic And Optimization Of The Photochemical Production Method Of Superoxide, Ta-Chung Ong

Honors Theses

The highly reactive nature of superoxide anion presents an analytical challenge to prepare stable standards for instrument calibration. McDowell et al. (1983) developed a convenient method of superoxide production based on continuous photolysis of benzophenone and acetone in oxygen saturated, alkaline, 2-propanol solution. In the McDowell reaction, ketones are photolyzed to their triplet state, and then reacted with alcohol to produce ketyl radicals that react with oxygen to produce superoxide. This study investigated the mechanism and rate of these reactions by Laser Flash Photolysis (LFP). Reaction rate constants for the reaction between the benzophenone triplet and ethanol was 5.5 x …

Intramolecular Chemistry Of Ss-Acetoxyethylidene And Photochemical Generation Of Vinylidenes, Jennifer M. Nguyen

Honors Theses

The parent carbene ß-acetoxyethylidene has been previously generated by the photolysis of its precursor, acetic acid 1a,9b-dihydro-1H-cyclopropa[l]phenanthren-1-yl methyl ester. Mechanistic studies on ß-acetoxyethylidene have indicated the possibilities of three rearrangement pathways to the formation of vinyl acetate: a proton shift, an acyl shift, and a cyclic rearrangement (see Figure 8). A deuterium labeling study involving the replacement of the hydrogens on the methylene carbon has indicated that the simple 1,2-H shift was the primary manner of rearrangement, with an 82% yield of product. However, a primary kinetic isotope effect may have occurred due to the greater strength of a C-D …

Physicochemical Properties Of Individual And Mixed Oxides Of Sio2, Al203, Tio2‚ And Photocatalytic Behaviour Of Organosilane Modified St Samples, Kalyani Gude

Masters Theses

A variety of oxides such as silica, alumina, titania and mixed oxides such as silica/alumina (SA), silica/titania (ST) and alumina/silica/titania (AST) are investigated by TGA and FTIR. These mixed oxides are analyzed because of their applications as catalysts. Thermogravimetric analysis is done on all the samples to determine the temperature at which the weakly adsorbed water is lost and the surface is left with only surface hydroxyls. TGA data showed the loss of most of the adsorbed water at around 100°C.

From the diffuse reflectance FTIR spectra taken at 200 and 300°C the silanol content is estimated from the 3740 …

Zinc(Ii) Incorporation Into Porphyrin Cytochrome C As A Function Of Temperature, Urea Concentration And Zinc(Ii) Acetate Concentration And The Urea And Guanidine Hydrochloride Induced Unfolding Of Porphyrin Cytochrome C, Sireesha Potula

Masters Theses

The objective of this thesis is to address the question of whether the removal of the native iron(II) ion in cytochrome c (cyt c) and substitution by a different metal ion affects the overall structural stability. We have studied the incorporation of zinc(II) into metal-free porphyrin cytochrome с (H2cyt) and investigated the structural stability of cyt c in the absence of the iron(II) ion and in the presence of the zinc(II) ion by different spectroscopic techniques. Compared to H2cyt, zinc-substituted cytochrome c (Zncyt) has a significantly different absorbance spectrum, so we monitor over time the change in absorbance upon addition …

Metal-Substituted Cytochrome C: Manganese (Ii) Incorporation Kinetics And Structural Stability Studies Of Native Iron Cytochrome C, Porphyrin Cytochrome C, Zinc Cytochrome C And Manganese Cytochrome C, Vineela Gundala

Masters Theses

Metalloproteins have the capacity to bind various metal ions. In spite of this seemingly diverse choice of cofactors, metalloproteins generally bind only their cognate metal in vivo. Cytochrome c (cyt c) binds Fe porphyrin in vivo. The main objective of this thesis is to investigate why nature selects Fe porphyrin as the prosthetic group in heme proteins and how removal of the native Fe in cyt c and substitution by different metal ions affects the local coordination environment, overall structural stability, and biological function. Our goal is to prepare and characterize metal-free cytochrome c, socalled porphyrin cytochrome c (H2cyt), and …

Coupling Coherence Distinguishes Structure Sensitivity In Protein Electron Transfer, Tatiana Prytkova, Igor V. Kurnikov, David Beratan

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Quantum mechanical analysis of electron tunneling in nine thermally fluctuating cytochrome b562 derivatives reveals two distinct protein-mediated coupling limits. A structure-insensitive regime arises for redox partners coupled through dynamically averaged multiple-coupling pathways (in seven of the nine derivatives) where heme-edge coupling leads to the multiple-pathway regime. A structure-dependent limit governs redox partners coupled through a dominant pathway (in two of the nine derivatives) where axial-ligand coupling generates the single-pathway limit and slower rates. This two-regime paradigm provides a unified description of electron transfer rates in 26 ruthenium-modified heme and blue-copper proteins, as well as in numerous photosynthetic proteins.

Use Of The Mitsunobu Reaction In The Synthesis Of Orthogonally Protected A,B-Diaminopropionic Acids, Fintan Kelleher, Keith Ó Proinsias

Articles

Orthogonally protected a,b-diaminopropionic acids have been synthesised in good yields by the reaction of N-trityl L-serine esters with N-substituted sulfonamides under Mitsunobu reaction conditions (DEAD, PPh₃, THF). The best isolated yields were obtained when N-Boc p-toluenesulfonamide was used as the nitrogen nucleophile precursor in the Mitsunobu reaction. Subsequently, the N-trityl group was efficiently replaced with the more stable allyloxycarbonyl (alloc) group.

Redox Potential Measurement In Aqueous Solutions And Biological Media, Mark M. Goldin, G. J. Blanchard, A. K. Evseev, V. A. Kolesnikov, Yu S. Goldfarb, A. G. Volkov, Mikhail M. Goldin

Faculty Publications and Presentations

No abstract provided.

Fast-Scan Cyclic Voltammetry - Scanning Electrochemical Microscopy, Luis Díaz-Ballote, Mario A. Apuche-Avlles, David O. Wipf

CAS Publications

The use of fast scan cyclic voltammetry (FSCV) to generate images in scanning electrochemical microscopy is demonstrated. In the FSCV-SECM method a cyclic voltammogram is acquired at each grid point of the scanned area. Thus, each point in the SECM image is a data array rather than a single value. This provides a significant increase in the amount of data collected per scan and provides the ability to make images of multiple analytes per scan. In addition, the use of cyclic voltammetry allows images based on adsorptive as well as diffusional processes. In this paper, we describe the FSCV-SECM experiment …

Single -Walled Carbon Nanotubes Separation And Redox Chemistry And Their Applications For Optical Biosensing And Scaffold Materials, Xiaomin Tu

Theses and Dissertations

Single-walled carbon nanotubes (SWNTs) have drawn great attention worldwide for their unique electronic, mechanical and optical properties. This dissertation details studies of some of the most important areas including: (1) Single-stranded DNA (ssDNA) assisted separation of SWNTs using high performance liquid chromatography; (2) Optical sensitivity and kinetic studies of redox reaction of ssDNA functionalized SWNTs with hydrogen peroxide (H 2 O 2 ); (3) Effects of Ag + on the redox reaction of ssDNA-encased SWNTs with hydrogen peroxide; (4) Single-walled carbon nanotube based optical sensors for DNA detection; and (5) SWNTs as scaffolds for promoting stem cell differentiation into bone …

Electrochemical Studies Of Some Furyl, Indol, Pyrrol, Hydroxynapthyl And Thienyl Derived Sulfonamides And Their Antibacterial Activities, Raja S. Machana

Theses and Dissertations

Sulfonamides are synthetic, wide-spectrum, antibiotics that find current therapeutic applications in the treatment of urinary tract infections, nocardiosis, and toxoplasmosis. Rapid development of bacterial resistance to this class of antimicrobial agents has obligated the need for synthesis of new sulfonamide derivatives. N 1 substitution with heterocyclic moieties produced variable antibacterial efficacies and their structure activity relationships were fairly established. However, para-amino (the N of which is designated as N 4 ) sulfonamide derivatives are less commonly studied. In this research we have selected a series of para-amino sulfonamide derivatives. Since redox potentials are important in determining the physiological activity of …

Activated Carbon Open Circuit Potential Shifts In Aqueous Solutions, Mikhail M. Goldin, Gary Blanchard, Alexander Volkov, Mogely Khubutiya, Vladimir Kolesnikov, Anatoly Evseev, Mark Goldin

Faculty Publications and Presentations

Interaction of certain organic compounds with activated carbon and its effect on the carbon open circuit potentials were studied. It was shown that shifts in open circuit potentials depended on the filling of the activated carbon surface. Whereas adsorption of the investigated compounds on the carbon led to positive potential shifts, their elimination (desorption) from carbon surface led to shifts in the opposite direction. It was also observed that the time dependence of potential shifts is similar for different carbon brands. The magnitude of shifts depended on adsorption activity and porometric characteristics of the carbon adsorbent, as well as the …