89 research outputs found

    Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

    Get PDF
    This work is licensed under a Creative Commons Attribution 4.0 International License.Free radical pathways play a major role in the degradation of protein pharmaceuticals. Inspired by biochemical reactions carried out by thiyl radicals in various enzymatic processes, this review focuses on the role of thiyl radicals in pharmaceutical protein degradation through hydrogen atom transfer, electron transfer, and addition reactions. These processes can lead to the epimerization of amino acids, as well as the formation of various cleavage products and cross-links. Examples are presented for human insulin, human and mouse growth hormone, and monoclonal antibodies

    Primary Processes of Free Radical Formation in Pharmaceutical Formulations of Therapeutic Proteins

    Get PDF
    Oxidation represents a major pathway for the chemical degradation of pharmaceutical formulations. Few specific details are available on the mechanisms that trigger oxidation reactions in these formulations, specifically with respect to the formation of free radicals. Hence, these mechanisms must be formulated based on information on impurities and stress factors resulting from manufacturing, transportation and storage. In more detail, this article focusses on autoxidation, metal-catalyzed oxidation, photo-degradation and radicals generated from cavitation as a result of mechanical stress. Emphasis is placed on probable rather than theoretically possible pathways

    Tyrosine Modifications in Aging

    Get PDF
    This is the publisher's version, also available electronically from http://online.liebertpub.com/doi/abs/10.1089/ars.2012.4595Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Recent Advances: Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3′-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Critical Issues: Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. Future Directions: The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind. Antioxid. Redox Signal. 17, 1571–1579

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

    Get PDF
    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

    Get PDF
    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

    Get PDF
    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

    Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase

    Get PDF
    AbstractMethionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR

    Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

    Get PDF
    Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cell but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad

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

    Get PDF
    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

    Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon beta-1a: potential implications for protein aggregation and immunogenicity

    Get PDF
    Oxidation via Cu2+/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu2+/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl) benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan and Tyr residues were identified throughout the primary sequence. Covalent crosslinks via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic
    • …
    corecore