Peptide cysteine thiyl radicals were generated through UV-photolysis of disulfide precursors, in order to follow intramolecular reactions of those radicals with neighboring amino acids. When reactions were carried out in D(2)O, there was a significant incorporation of deuterium specifically into the C(alpha)-H bonds of glycine residues in positions i+1 and i-1 to the Cys residue, indicating a fast reversible H-atom transfer. This H-atom transfer occurred prior to the formation of final, nonradical products including free thiol, thioaldehyde, and aldehyde. Such fast H-atom transfer is relevant to biologic conditions of oxidative stress and to the stabilization of proteins against oxidation, where the formation of carbon-centered radicals in proteins may lead to fragmentation, intramolecular cross-linking, aggregation and/or epimerization.

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.

The selective oxidative modification of proteins can have significant consequences for structure and function. Here, we show that protein cysteine thiyl radicals (CysS*) can reversibly abstract hydrogen atoms from the alpha C-H bonds of selected amino acids in a protein (insulin). CysS* were generated photolytically through homolysis of cystine and through photoionization of an aromatic residue, followed by one-electron reduction of cystine. Intramolecular hydrogen transfer was monitored through the covalent incorporation of deuterium into specific amino residues. Of 51 insulin amino residues, only six incorporated significant levels of deuterium: Leu(B6), Gly(B8), Ser(B9), Val(B18), Gly(B20), and Cys(A20). All these amino acids are located at the beginning/end or outside of alpha-helices and beta-sheets, in accordance with theory, which predicts that specifically the alpha C-H bonds of amino acids in these secondary structures have higher homolytic C-H bond dissociation energies compared to the alpha C-H bonds of amino acids in extended conformations. Through such hydrogen transfer mechanisms, thiyl radicals are able to catalyze the oxidation of amino acids in a protein through oxidants, which would not necessary directly react with these amino acids. This feature has important consequences for protein stability under conditions of oxidative stress and/or protein production in pharmaceutical biotechnology.

The photodissociation of intrachain disulfide bonds in a model peptide and salmon calcitonin generates a series of cyclic peptide products following the generation of a CysS(*) thiyl radical pair. Key to the formation of these cyclic products are disproportionation and reversible hydrogen atom transfer reactions as well as secondary photoreactions, which lead to C-S bond breakage of primary photoproducts. Depending on the wavelength of the incident light, disulfides ultimately convert into cyclic thioethers. An important photolytic product is H(2)S, which is highly relevant for the production and storage of protein pharmaceuticals, where H(2)S can catalyze disulfide scrambling and protein degradation.

Recently, we characterized a thiyl radical-dependent mechanism for the photolytic conversion of a disulfide bond in a model peptide into dithiohemiacetal and subsequently into thioether ( Mozziconacci et al. ( 2010 ) J. Phys. Chem B 114 , 3668 - 3688 ). This mechanism is of potential relevance for the photodegradation of disulfide-containing proteins, which may be a problem for the production and formulation of diagnostic and therapeutic protein pharmaceuticals. In this Rapid Report, we show that similar products are also formed when an antibody (IgG1) is subjected to photoirradiation at 253.7 nm, suggesting the involvement of thiyl radicals also in these processes. A series of dithiohemiacetal and thioether cross-links were identified in photoirradiated IgG1 through HPLC-MS/MS analysis.

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 highconcentration 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 istaroximetreated 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. Clinical RelevanceThis study defines impact of inhalation of a toxic gas, chlorine, on a critical cardiac calcium pump, sarcoendoplasmic reticulum Ca 21 ATPase (SERCA). It also demonstrates that therapeutic strategies that protect or modify SERCA function could be useful approaches for emergent resuscitation of severe chlorine inhalation victims.Chlorine is a commonly used chemical in industry and society. Acute chlorine inhalation toxicity can occur due to accidents at swimming pools and/or involving water purification systems, after transportation accidents, upon industrial exposure, with misuse of domestic cleaners, during military operations, and, more recently, through chemical terrorism. In the

The reversible intramolecular hydrogen transfer reaction of peptide Cys thiyl radicals with Gly and Ala residues was studied in model peptides, where thiyl radicals were either generated through photochemical cleavage of disulfide bonds or through the reaction of Cys thiol with (*)CH(3) or CH(3)C(*)O radicals, or both, generated through photolysis of acetone. In D(2)O, the reversible hydrogen transfer leads to covalent H/D exchange, indicative of the location of intermediary carbon-centered radicals. In addition, the reversible formation of (alpha)C(*) radicals on Ala leads to the conversion of L-Ala to D-Ala, where the efficiency of this conversion depends on the primary sequence of the Ala-containing peptide. When Cys thiyl radicals are generated through the reaction of Cys thiol with (*)CH(3) or CH(3)C(*)O radicals, various recombination products between these initiating radicals and peptide thiyl and carbon-centered radicals provide further evidence for the location of intermediary radicals within the peptide sequence.

PurposeTo elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).MethodsInsulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin.ResultsThe results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected.ConclusionMCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.