Research and Publications - Department of Chemistry & Biochemistry
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Item Polybenzocrown ethers: synthesis by cesium-assisted cyclization and solid-state structures(ARKAT USA, Inc., 2010-09-05) Hanes, Robert E., Jr.; Ellingsworth, Edward C.; Griffin, Scott T.; Rogers, Robin D.; Bartsch, Richard A.A series of large-ring polybenzocrown ethers is prepared by cesium-assisted cyclizations. Reactions of diphenols/bisphenols, dimesylates of oligoethylene glycols and cesium carbonate in MeCN produce the large-ring polybenzocrown ethers in high yields. To gain further insight into the structures of these compounds, solid-state structures of three large-ring crown ethers are obtained by X-ray diffraction.Item Solvent-free synthesis of benzothiazole-based quaternary ammonium salts: precursors to ionic liquids(ARKAT USA, Inc., 2010-04-20) Nadeem, Sohail; Munawar, Munawar A.; Ahmad, Saeed; Smiglak, Marcin; Drab, David M.; Malik, Khizar I.; Amjad, Rana; Ashraf, Chaudry M.; Rogers, Robin D.A series of 13 benzothiazolium iodide salts have been prepared in solvent-free conditions by optimizing the reaction. An additional 26 benzothiazolium salts with bistrifluoromethanesulfonimide and trifluoromethylsulfonate were prepared via simple metathesis reactions from the iodide precursors. Out of a total of 39 prepared salts, 26 were identified as ionic liquids, with melting points as low as 42 degrees Celsius observed for dodecylbenzothiazolium bistrifluoromethanesulfonimide. All prepared compounds have been characterized using FTIR, 1H, 13C NMR, and HRMS analyses. The thermal stabilities as well as melting points of these salts were also analyzed.Item C-methylation occurs during the biosynthesis of heme d1(American Society for Biochemistry and Molecular Biology, 1990-08-15) Yap-Bondoc, Flordeliza; Bondoc, Laureano L.; Timkovich, Russell; Baker, David C.; Hebbler, AnnaThe biosynthetic origin of methyl groups in heme d1 isolated from the nitrite reductase cytochrome cd1 was investigated by a stable isotope labeling experiment. Pseudomonas aeruginosa (American Type Culture Collection strain 19429) was grown on a minimal medium supplemented with [13C]methionine. The enzyme was purified, the heme extracted, converted into the free base methyl ester derivative, and purified. 1H NMR and 13C NMR indicated that only the methyl groups attached to C2 and C7 are derived from methionine.Item Inactivation of cytochrome cd1 by hydrazines(American Society for Biochemistry and Molecular Biology, 1990-03-15) Yap-Bondoc, Flordeliza; Timkovich, RussellThe dissimilatory nitrite reductase, cytochrome cd1, from Pseudomonas aeruginosa (ATCC 19429) was irreversibly inactivated by methyl- or phenylhydrazine but was only reduced by hydrazine itself. The reaction required oxygen and several turnovers, approximately four, of the cytochrome acting to transfer reducing equivalents from phenylhydrazine to oxygen. The reaction with methyl- or phenylhydrazine altered the visible spectrum of the cytochrome. Bands characteristic of reduced heme c appeared plus new features that were not characteristic of either oxidized or reduced heme d1. Extraction of the heme from phenylhydrazine-treated cytochrome yielded a covalently modified form of the original heme d1. Visible, 1H NMR, and mass spectra were obtained on the purified modified heme and on the metal-free esterified derivative. The spectroscopic data indicate that the modification was the regiospecific substitution of the 5 meso-proton by a phenyl group.Item Non-B right-handed DNA conformations of homopurine.homopyrimidine sequences in the murine immunoglobulin C alpha switch region(American Society for Biochemistry and Molecular Biology, 1988-05-25) Collier, David A.; Griffin, Johanna A.; Wells, Robert D.The switch region of IgA immunoglobulin in mice cloned into a recombinant plasmid contains a supercoil-dependent S1 nuclease hypersensitive site, indicative of a non-B-DNA secondary structure. This site maps to the (AGGAG)28 direct repeat (DR2) of the alpha switch region and appears at a negative superhelical density of greater than 0.02. Studies with P1 nuclease and bromoacetaldehyde indicate that this structure is also present at neutral pH. S1 nuclease sensitivity is retained for the shorter repeat (AGGAG)6GA in a recombinant plasmid but is not seen for the repeat (CTGAG)6, corresponding to the DR1 repeat of the alpha switch region, or in a sequence corresponding to a portion of the consensus sequence which contains a short stretch of alternating pyrine-pyrimidine residues. Fine mapping of the (AGGAG)6GA and flanking sequences with dimethyl sulfate, bromoacetaldehyde, osmium tetroxide, and diethyl pyrocarbonate reveals an asymmetric pattern of modification dependent on both pH and supercoiling. Two-dimensional gel electrophoresis at low pH shows the relaxation of 3 superhelical turns on formation of this structure by the (AGGAG)6GA repeat. These results are most consistent with the formation of an intramolecular triple-strand.Item In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha-subunits(American Society for Biochemistry and Molecular Biology, 1986-12-15) Milton, Debra L.; Napier, Mary L.; Myers, Richard M.; Hardman, John K.A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan synthase. A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the alpha-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagenized in vitro (using nitrous acid, formic acid, hydrazine, and potassium permanganate), and several thousands of mutants have been isolated. Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single amino acid substitutions. Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by lactose. The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein. A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures. The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed. Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha-subunit. An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many amino acid alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide.Item 2-Methylene-13-dicelenole: A precursor of tetraselenafulvalene (TSeF)(Elsevier, 1987)The preparation of methylene-1, 3-diselenole and its ready conversion to TSeF are discussed. The preparation of methylene-1, 3-diselenole and its ready conversion to tetraselenafulvalene (TSeF) are described.Item Disassembly and Degradation of Photosystem I in an in Vitro System Are Multievent, Metal-dependent Processes(American Society for Biochemistry and Molecular Biology, 2003-10) Henderson, J. Nathan; Zhang, Jianying; Evans, B. Walter; Redding, KevinAn in vitro system was created to study the process of membrane protein degradation by using photosystem I (PS1) as a model membrane protein. Purified chloroplast membranes were incubated at 30 °C in a defined buffer along with various extracts or reagents to reconstitute the disassembly and degradation of PS1, which was monitored by a variety of techniques that probe the integrity of the PS1 complex: photo-biochemical assays, semi-native gel electrophoresis, low temperature fluorescence spectroscopy, and immunoblots using antibodies against different PS1 subunits. During a typical time course, degradation of PS1 appeared to be a multievent process, with disassembly of the complex preceding proteolysis of the subunits. The first change seen was a rapid (<5 min) decrease in PS1 photochemical activity. This was followed by a diminution of far-red fluorescence emission from the core antenna of PS1 and a slower disassembly of the PS1 chlorophyll-protein core complex, as visualized by semi-native gel electrophoresis. Surprisingly, the latter was not accompanied by a similar rate of proteolysis of the PsaA core subunit. In contrast, addition of soluble proteases caused rapid loss of immuno-detectable PS1 polypeptides and cleavage of the major PS1 polypeptides in interhelical loops. The in vitro degradation process was time- and temperature-dependent but did not require ATP, GTP, or soluble chloroplast proteins. Chelation of divalent cations by EDTA inhibited the later steps of disassembly and proteolysis, and this effect could be reversed by addition of micromolar Zn2+, with Co2+ and Ca2+ providing somewhat lower activity.Item Electron Flow between Photosystem II and Oxygen in Chloroplasts of Photosystem I-deficient Algae Is Mediated by a Quinol Oxidase Involved in Chlororespiration(American Society for Biochemistry and Molecular Biology, 2000-06-09) Cournac, Laurent; Redding, Kevin; Ravenel, Jacques; Rumeau, Dominique; Josse, Eve-Marie; Kuntz, Marcel; Peltier, GillesIn Chlamydomonas reinhardtii mutants deficient in photosystem I because of inactivation of the chloroplast genes psaA or psaB, oxygen evolution from photosystem II occurs at significant rates and is coupled to a stimulation of oxygen uptake. Both activities can be simultaneously monitored by continuous mass spectrometry in the presence of18O2. The light-driven O2 exchange was shown to involve the plastoquinone pool as an electron carrier, but not cytochrome b 6 f. Photosystem II-dependent O2 production and O2 uptake were observed in isolated chloroplast fractions. Photosystem II-dependent oxygen exchange was insensitive to a variety of inhibitors (azide, carbon monoxide, cyanide, antimycin A, and salicylhydroxamic acid) and radical scavengers. It was, however, sensitive to propyl gallate. From inhibitors effects and electronic requirements of the O2 uptake process, we conclude that an oxidase catalyzing oxidation of plastoquinol and reduction of oxygen to water is present in thylakoid membranes. From the sensitivity of flash-induced O2 exchange to propyl gallate, we conclude that this oxidase is involved in chlororespiration. Clues to the identity of the protein implied in this process are given by pharmacological and immunological similarities with a protein (IMMUTANS) identified in Arabidopsis chloroplasts.Item Structural Characterization of Nitrimyoglobin(American Society for Biochemistry and Molecular Biology, 1989-04-15) Bondoc, Laureano L.; Timkovich, RussellNitrimyoglobin was formed in greater than 94% yield by a simple reaction between excess nitrite and horse heart metmyoglobin at pH 5.5. This dark green pigment was shown by 1H NMR spectroscopy to be a single, pure product with a well defined tertiary structure that is highly similar to the starting myoglobin. Electronic spin states parallel those of myoglobin, although the relaxation times differ. Ligand binding reactions of nitrimyoglobin parallel those of normal myoglobin, but lead to a unique series of UV-visible spectra. In the ferrous state, nitrimyoglobin reversibly binds O2 with half-saturation of sites at an O2 partial pressure of 10.4 ± 1.4 mm Hg. 1H NMR data indicate that the altered heme of nitrimyoglobin has not undergone reaction at any meso proton position, nor has it been partially saturated to the level of a chlorin. 15N NMR spectra indicate that only a single nitrogen was added to the protein as a nitro group. Extraction of the modified heme from nitrimyoglobin and spectroscopic characterization of the nitriheme by infrared spectroscopy and of the free base porphyrin methyl ester derived from nitriheme by 1H NMR indicate that the modification is regiospecific. The heme in nitrimyoglobin is 3-(trans-2-nitrovinyl)-2,7,12,18-tetramethyl-8-vinylporphyrin-13,17-dipropionic acid. In the Fisher nomenclature scheme, the 2-vinyl substituent is the site of modification and has been converted to a nitrovinyl group by substitution of a proton by -NO2.Item Radical reactions involving cyclodextrins and model sugars(UMI Company, 1997) Lehmann, Marc N.Time-resolved EPR/laser flash photolysis studies of aqueous solutions of D-glucose, alpha-methyl-D-glucose, maltose and alpha-, beta- and gamma-cyclodextrin (CD) with acetone, pyruvic acid (PA), methylethylketone (MEK) and levulinic acid (LA) showed that the excited triplet state of these ketones could be used to selectively produce different polarized sugar radicals depending on the substrate used. For both D-glucose and maltose the C1 carbon centered radical was observed to be most intense and thought to be a consequence of the anomeric effect which favors binding of the ketone to the hydroxyl group situated on this carbon. For alpha-methyl-D-glucose the C7, C6 and C2 centered radicals were most intense and their selective formation is attributed to affinity effects. The following CD radicals were identified. From alpha-CD; C3 and C5 centered radicals. From beta-CD; C3, C5 and C1. From gamma-CD; C1 and C6. A C5 carbon centered radical formed from glucopyranoside ring-opening of the C1 radical was also observed for beta-CD and gamma-CD. Changes in the intensity of the spectra of the radicals observed between alpha-, beta- and gamma-CD were explained to arise from the extent ketone binding to the cyclodextrin cavity.Item Tryptophan operon read-through. Isolation and characterization of an abnormally long tryptophan synthetase alpha subunit from a frame-shift mutant of Escherichia coli(American Society for Biochemistry and Molecular Biology, 1975-06-25) Hardman, John K.; Berger, Hillard; Goodman, MyronA new mutant strain of Escherichia coli, strain ICR-47, contains a frame-shift mutation in the trpA gene, the gene most distal to the operator in the trp operon. Mapping experiments indicate that the lesion is located at a site within 10 to 15% of the end of this gene. The mutation results in "out-of-phase" translation of the distal portion of the trp mRNA; normal translational termination signal(s) are not encountered and a trpA gene product longer than the wild type protein is produced. As with the other enzymes produced from this operon, the in vivo level of the altered protein (the alpha subunit of the tryptophan synthetase enzyme complex) is controlled by exogenous L-tryptophan. The altered alpha subunit from the strain ICR-47 has been isolated and characterized. Molecular weight estimations indicate a molecular weight of approximately 37,000, an increase beyond the wild type enzyme corresponding to an additional 50 to 70 amino acid residues. The protein has a new COOH-terminal amino acid sequence. Results of preliminary hybridization experiments suggest that the ICR-47 mRNA, which is necessarily longer than that needed to code for wild type enzyme, is not detectably different in size from wild type mRNA. The enzymatic properties of the ICR-47 alpha subunit indicates a greatly reduced ability of the mutant subunit to combine functionally with wild type beta2 subunit, the second protein component in the tryptophan synthetase enzyme complex. In contrast, only 40 to 50% of the intrinsic enzymatic activity of the alpha subunit is lost.Item Constituents of the Blood of the Hibernating and Normal Rattlesnake, Crotalus Horridus(1945-12-01) Carmichael, Emmett B.; Petcher, Paul W.Item William Owen Baldwin(Paul B. Hoeber, 1942) Carmichael, Emmett B.Item La Fayette Guild(Paul B. Hoeber, 1935-03) Carmichael, Emmett B.Item Charles Alexander Pope(P.B. Hoeber, 1940-09-01) Carmichael, Emmett B.Item On Osmious Acid, and the Position of Osmium in the List of Elements(American Journal of Science, 1860) Mallet, John WilliamsItem Theory of electrical rectification in a molecular monolayer(American Physical Society, 2001) Krzeminski, C.; Delerue, C.; Allan, G.; Vuillaume, D.; Metzger, R.M.The current-voltage characteristics in Langmuir-Blodgett monolayers of γ-hexadecylquinolinium tricyanoquinodimethanide (C16H33Q-3CNQ) sandwiched between Al or Au electrodes is calculated, combining ab initio and self-consistent tight binding techniques. The rectification current depends on the position of the LUMO and HOMO relative to the Fermi levels of the electrodes as in the Aviram-Ratner mechanism, but also on the profile of the electrostatic potential which is extremely sensitive to where the electroactive part of the molecule lies in the monolayer. This second effect can produce rectification in the direction opposite to the Aviram-Ratner prediction.Item Evidence for two active branches for electron transfer in photosystem I(The National Academy of Sciences, 2001) Guergova-Kuras, Mariana; Boudreaux, Brent; Joliot, Anne; Joliot, Pierre; Redding, KevinAll photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.Item Synthesis and Characterization of Multifunctional Chitosan- MnFe2O4 Nanoparticles for Magnetic Hyperthermia and Drug Delivery(MDPI, 2010) Kim, Dong-Hyun; Nikles, David E.; Brazel, Christopher S.; University of Alabama TuscaloosaMultifunctional nanoparticles composed of MnFe2O4 were encapsulated in chitosan for investigation of system to combine magnetically-triggered drug delivery and localized hyperthermia for cancer treatment with the previously published capacity of MnFe2O4 to be used as an efficient MRI contrast agent for cancer diagnosis. This paper focuses on the synthesis and characterization of magnetic MnFe2O4 nanoparticles, their dispersion in water and their incorporation in chitosan, which serves as a drug carrier. The surface of the MnFe2O4 nanoparticles was modified with meso-2,3-di-mercaptosuccinic acid (DMSA) to develop stable aqueous dispersions. The nanoparticles were coated with chitosan, and the magnetic properties, heat generation and hydrodynamic size of chitosan-coated MnFe2O4 were evaluated for various linker concentrations and in a range of pH conditions.