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The reaction pathways of hydrogen peroxide in the presence of scavengers and its transition metal catalyzed oxidation of disulfide bridge containing peptides
Asmus, Christopher Erik Helmut
Asmus, Christopher Erik Helmut
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Abstract
The degradation pathway of hydrogen peroxide (H2O2) with sodium pyruvate elucidates the formation and breakdown of an intermediate formed during the reaction. Sodium pyruvate is a potential base for the creation of a peroxide scavenger. It readily reacts with H2O2 to yield sodium acetate, carbon dioxide and water as final products. A low temperature nuclear magnetic resonance protocol has been developed in order to observe the formation and breakdown of a proposed intermediate, 2-hydroxy-hydroperoxy-propanoate. Samples of 13C-enriched sodium pyruvate were used to verify the formation of the intermediate and identify its structure by the heteronuclear multiband correlation spectroscopy NMR method. The observed chemical shifts have been compared with the predicted chemical shifts obtained from density functional theory calculations. In order to calculate the activation parameters of the formation and breakdown of the intermediate a low temperature 13C-NMR protocol has been developed that allowed to record intergable spectra of sodium pyruvate, the intermediate and the products formed by the reaction with H2O2 at various temperatures. The integrated data have been plotted vs. time which allowed to calculate the individual rate constants of the reaction steps at various temperatures. Based on the rate constants the activation parameters, the enthalpy, entropy and free energy of the transition states of the formation and breakdown of the intermediate have been calculated. The metal-catalyzed pathway of hydrogen peroxide is dealing with the effect of hydroxyl radicals created by the Fenton reaction and their potential to oxidize the disulfide bridge of small peptides. Based on the reduction potential, which is an indicator for the oxidizability by radicals, disulfide bridges in a peptide or protein are more facile targets for radical attack than methionine (Met) which is known to be easily oxidized by reactive oxygen species. The effects of the Fenton reaction on the peptides pressinoic acid (PA), oxytocin (Oxt) and a custom made peptide containing a disulfide bridge have been studied. The experiments showed that the aromatic residues tyrosine (Tyr), which present on both PA and Oxt and Phenylalanine (Phe), which occurs on PA as well as arginine (Arg) located on the custom made peptide have been subject to hydroxylation by the ROS created through the Fenton reaction. No modification of the disulfide bridge such as oxidation, disulfide scrambling or formation of new carbon-sulfur bonds was detected on these peptides after the Fenton reaction has been applied.
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Date
2015-05-31
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University of Kansas
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Keywords
Chemistry, Pharmaceutical sciences, Analytic, hydroxyphenylalanine, Intermediate, NMR