Reversible hydrogen transfer reactions of cysteine thiyl radicals in peptides: the conversion of cysteine into dehydroalanine and alanine, and of alanine into dehydroalanine

The photodissociation of disulfide bonds in model peptides containing Ala and Ala-d3 generates a series of photoproducts following the generation of a CysS• thiyl radical pair. These photoproducts include transformations of Cys to dehydroalanine (Dha) and Ala, as well as Ala to Dha. Intramolecular Michael addition of an intact Cys with a photolytically generated Dha results in the formation of cyclic thioethers. The conversion of Cys into Dha likely involves a 1,3-H-shift from the Cys αC-H bond to the thiyl radical, followed by elimination of HS•. The conversion of Dha into Ala most likely involves hydrated electrons, which are generated through the photolysis of Cys, the photoproduct of disulfide photolysis. Prior to stable product formation, CysS• radicals engage in reversible hydrogen transfer reactions with αC-H and βC-H bonds of the surrounding amino acids. Especially for the βC-H bonds of Ala such hydrogen transfer reactions are unexpected based on thermodynamic grounds; however, the replacement of deuterons in Ala-d3 by hydrogens in H2O provides strong experimental evidence for such reactions

Low-Temperature NMR Characterization of Reaction of Sodium Pyruvate with Hydrogen Peroxide

It was proposed that the reaction of sodium pyruvate and H2O2 generates the intermediate 2-hydroperoxy-2-hydroxypropanoate, which converts into acetate, CO2, and H2O (Aleksankin et al. Kernenergie 1962, 5, 362–365). These conclusions were based on the products generated in 18O-enriched water and H2O2 reacting with pyruvic acid at room temperature; however, the lifetime of 2-hydroperoxy-2-hydroxypropanoate at room temperature is too short for direct spectroscopic observation. Therefore, we applied the combination of low-temperature and 13C NMR techniques to verify, for the first time, the formation of 2-deuteroperoxy-2-deuteroxypropanoate in mixtures of D2O and methanol-d4 and to monitor directly each species involved in the reaction between D2O2 and 13C-enriched pyruvate. Our NMR results confirm the formation of 2-deuteroperoxy-2-deuteroxypropanoate, where the respective chemical shifts are supported by density functional theory (DFT) calculations. At near-neutral apparent pD (pD*) and −35 °C, the formation of 2-deuteroperoxy-2-deuteroxypropanoate occurred with k = 2.43 × 10−3 dm3·mol−1·s−1. The subsequent decomposition of 2-deuteroperoxy-2-deuteroxypropanoate into acetate, CO2, and D2O occurred with k = 2.58 × 10−4 s−1 at −35 °C. In order to provide a full kinetic analysis, we also monitored the equilibrium of pyruvate and methanol with the hemiacetal (2-deuteroxy-2-methoxypropanoate). The kinetics for the reaction of sodium pyruvate and D2O2 were fitted by taking into account all these equilibria and species

Metal-Catalyzed Oxidation of Protein Methionine Residues in Human Parathyroid Hormone (1-34): Formation of Homocysteine and a Novel Methionine-Dependent Hydrolysis Reaction

The oxidation of PTH(1-34) catalyzed by ferrous ethylenediaminetetraacetic acid (EDTA) is site-specific. The oxidation of PTH(1-34) is localized primarily to the residues Met[8] and His[9]. Beyond the transformation of Met[8] and His[9] into methionine sulfoxide and 2-oxo-histidine, respectively, we observed a hydrolytic cleavage between Met[8] and His[9]. This hydrolysis requires the presence of FeII and oxygen and can be prevented by diethylenetriaminepentaacetic acid (DTPA) and phosphate buffer. Conditions leading to this site-specific hydrolysis also promote the transformation of Met[8] into homocysteine, indicating that the hydrolysis and transformation of homocysteine may proceed through a common intermediate

Light-induced Conversion of Trp to Gly and Gly Hydroperoxide in IgG1

The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by αC-βC cleavage of the Trp side chain. The resulting glycyl radicals are either reduced to Gly, or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photo-induced electron- or hydrogen-transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals

Intramolecular hydrogen transfer reactions of thiyl radicals from glutathione: formation of carbon-centered radical at Glu, Cys and Gly

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3000494Glutathione thiyl radicals (GS•) were generated in H2O and D2O by either exposure of GSH to AAPH#, photoirradiation of GSH in the presence of acetone, or photoirradiation of GSSG. Detailed interpretation of the fragmentation pathways of deuterated GSH and GSH-derivatives during mass spectrometry analysis allowed us to demonstrate that reversible intramolecular H-atom transfer reactions between GS• and C-H bonds at Cys[αC], Cys[βC], and Gly[αC] are possible

Do not drop: Mechanical shock in vials causes cavitation, protein aggregation and particle formation

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond timecourse. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues

Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.As the second of a 3-part series of articles in this issue concerning the development of a mathematical model for comparative characterization of complex mixture drugs using crofelemer (CF) as a model compound, this work focuses on the evaluation of the chemical stability profile of CF. CF is a biopolymer containing a mixture of proanthocyanidin oligomers which are primarily composed of gallocatechin with a small contribution from catechin. CF extracted from drug product was subjected to molecular weight–based fractionation and thiolysis. Temperature stress and metal-catalyzed oxidation were selected for accelerated and forced degradation studies. Stressed CF samples were size fractionated, thiolyzed, and analyzed with a combination of negative-ion electrospray ionization mass spectrometry (ESI-MS) and reversed-phase-HPLC with UV absorption and fluorescence detection. We further analyzed the chemical stability data sets for various CF samples generated from reversed-phase-HPLC-UV and ESI-MS using data-mining and machine learning approaches. In particular, calculations based on mutual information of over 800,000 data points in the ESI-MS analytical data set revealed specific CF cleavage and degradation products that were differentially generated under specific storage/degradation conditions, which were not initially identified using traditional analysis of the ESI-MS results

Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.This work was supported by the CounterACT Program, National Institutes of Health, Office of the Director, and the National Institute of Environmental Health Sciences grant U54 ES015678 (C.W.W.), and by Children’s Hospital of Colorado/Colorado School of Mines Pilot Award G0100394 and a Children’s Hospital of Colorado Research Institute’s Pilot Award (S.A.)