42 research outputs found
Stepwise O atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening
This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/jacs.8b00262.Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV–vis spectroscopy, high-resolution mass spectrometry, 1H NMR, 13C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases
An overall evaluation of the resistance (R) and pathogenesis-related (PR) superfamilies in soybean, as compared with Medicago and Arabidopsis
Studies in uric acid metabolism in man : variations in the uric acid content of normal human saliva
Thesis (M.S.)--University of Illinois, 1919.Typescript.Includes bibliographical references (leaf 26)
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Development of a mass spectrometry-based method for studying hemoglobin assembly/disassembly
A mass spectrometry-based method was developed for studying hemoglobin assembly/dissociation. The dynamics of bovine hemoglobin assembly were investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. The mass spectral data confirm that bovine hemoglobin dissociation involves a step where heme is first lost from the β-chain of the α*β*-dimer to form a heme-deficient dimeric species or semi-hemoglobin dimer (α*β*). The experimental data provide strong evidence that binding of a partially unstructured apo-β-chain to a tightly folded holo-α-chain to form the semi-hemoglobin dimer is the initial step of hemoglobin assembly. Such binding locks the β-chain in a highly ordered conformation, which allows for an efficient heme acquisition. This step is followed by the docking of the two hemoglobin dimers to form a tetrameric form of the protein. Also, investigation of the individual apo- and heme-reconstituted globin chains show that although apo-α- and apo-β-chains are very flexible, dynamic, and have similar protein structures (the globin fold), reconstituted α-chains are monomeric and exhibit conformational structures very similar to holo-α-chains in the complex Hb equilibrium mixture. Reconstituted β-chains oligomerize to form the tetrameric homo-β-globin species HbH found in alpha thalassemic disorder. This suggests that dynamics play an important, perhaps the most important, role in the hemoglobin assembly process. We hypothesize that the intrinsic protein disorder exhibited by the bovine hemoglobin β-chains and the asymmetry of globin interaction reported in this work developed due to evolutionary pressure to provide a vital safeguard that may inhibit random oligomerization and aggregation in the crowded environment of the red blood cell, thus directing the assembly process along the correct pathway. The data support this hypothesis as hemoglobins analyzed from all organisms that evolved after the emergence of separate α- and β-chains (bovine, porcine, human, and modern fish hemoglobins) exhibit the expected asymmetry in the roles played by their globin chains in assembly. In contrast, hemoglobin from the mollusk Scapharca, which parallels the emergence of separate α- and β-chains does not exhibit asymmetry. In the analysis of the modern fish hemoglobin from Gadus morhua, the Atlantic cod, it was noticed that the measured molecular weights for the α- and β-chains did not correspond to masses calculated from their published sequences. Using mass spectrometry-based methods 65% sequence coverage was achieved, and a number of mutations were identified in both globin chains. A feasibility study for the use of mass spectrometry to study the interaction between hemoglobin and its scavenger protein haptoglobin is also presented
Highly Asymmetric Interactions between Globin Chains during Hemoglobin Assembly Revealed by Electrospray Ionization Mass Spectrometry â€
Hemoglobin redux: combining neutron and X-ray diffraction with mass spectrometry to analyse the quaternary state of oxidized hemoglobins
X-ray and neutron diffraction studies of cyanomethemoglobin are being used to evaluate the structural waters within the dimer–dimer interface involved in quaternary-state transitions
Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine
Mass Spectrometric Identification of Silver Nanoparticles: The Case of Ag<sub>32</sub>(SG)<sub>19</sub>
Mass spectrometry has played a key role in identifying
the members
of a series of gold clusters, which has enabled the development of
magic-number cluster theory. The successes of the gold cluster system
have yet to be repeated in another metal cluster system, however.
Silver clusters in particular have proven to be challenging due to
their relative instability compared with gold clusters. Using the
well-characterized gold nanocluster, Au<sub>25</sub>(SG)<sub>18</sub>, we present optimized electrospray ionization mass spectrometry
(ESI-MS) instrumental parameters for the maximal transmission of the
intact cluster. Parameters shown to have the largest effect on intact
cluster transmission/detection include trap and transfer collision
energy, source temperature, and cone gas flow rate. Herein we describe
a general strategy to acquire mass spectra of fragile metal clusters
with reliable mass assignments. By also optimizing sample solution
conditions, high-quality ESI mass spectra of a prototypical silver:glutathione
(Ag:SG) cluster were obtained without significant fragmentation. By
using gentle conditions and solution conditions designed to stabilize
the clusters, fragmentation was dramatically reduced and mass spectra
with isotopic resolution were measured. Using this strategy, we have
made the first formula assignment for a ligand-protected Ag cluster
of Ag<sub>32</sub>(SG)<sub>19</sub>
M<sub>3</sub>Ag<sub>17</sub>(SPh)<sub>12</sub> Nanoparticles and Their Structure Prediction
Although
silver nanoparticles are of great fundamental and practical
interest, only one structure has been determined thus far: M<sub>4</sub>Ag<sub>44</sub>(SPh)<sub>30</sub>, where M is a monocation, and SPh
is an aromatic thiolate ligand. This is in part due to the fact that
no other molecular silver nanoparticles have been synthesized with
aromatic thiolate ligands. Here we report the synthesis of M<sub>3</sub>Ag<sub>17</sub>(4-<i>tert</i>-butylbenzene-thiol)<sub>12</sub>, which has good stability and an unusual optical spectrum. We also
present a rational strategy for predicting the structure of this molecule.
First-principles calculations support the structural model, predict
a HOMO–LUMO energy gap of 1.77 eV, and predict a new “monomer
mount” capping motif, AgÂ(SR)<sub>3</sub>, for Ag nanoparticles.
The calculated optical absorption spectrum is in good correspondence
with the measured spectrum. Heteroatom substitution was also used
as a structural probe. First-principles calculations based on the
structural model predicted a strong preference for a single Au atom
substitution in agreement with experiment