Characterisation of the Novel Products of Protein Oxidation by the Inflammatory Oxidant Hypochlorous Acid (HOCl) Using Raman Spectroscopy

classical cell death pathway which features nuclear condensation and mitochondrial damage, accompanied by disruptions to cellular bioenergetics and loss of ATP. Analysis with the membrane probe DiI revealed that the helicate also initiates re-organization of membrane structure, suggesting that [Pd2(hextrz)4]4+ initially interacts with lipids to disrupt cellular compartmentalization, followed by changes to metal ion homeostasis and organelle function. These data provide the first indication that quadruply stranded helicates interact with cells via a different mechanism to other supramolecular complexes, raising the possibility that they may become therapeutic alternatives to current cytotoxic agents.

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doi: 10.1016/j.freeradbiomed.2016.10.056

Nitric oxide (NO) is sensitively and unambiguously detected by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, EPR can identify the nitrogenous substrate of NO production using the stable isotope 15N. However, the dithiocarbamate-iron spin traps employed with this method have limited its application in biological samples. These limitations were overcome using a liposome-encapsulated spin-trap (LEST) that is easily prepared. This method can detect as little as 40 nM NO in a 500 µL sample (≥ 20 pmol). Application of the LEST method to cell lysates of the coccolithophore Emiliania huxleyi allowed for the detection of biological NO production from as little 17 µg of total protein.

16 Human Lung Disease-Associated Proteins Are Oxidized in Mouse Lung by Cadmium Levels Found in Human Environmental and Dietary Exposures Young-Mi Go1, Joshua D. Chandler1, Michael Orr1, Yongliang Liang1, and Dean P. Jones1 1 Emory University, Atlanta, United States Cd accumulates in humans from dietary, environmental including cigarette smoke, and occupational sources, and has a twenty-year biologic half-life. Our previous studies show that environmental low dose Cd oxidizes protein redox states and stimulates inflammatory signaling and actin cytoskeleton disruption; these results suggest that Cd could impact multiple disease mechanisms. Although considerable information is available concerning effects of high dose occupational Cd exposures and cigarette smoking, little is known about the relative sensitivities of subcellular compartmental redox proteome and the effect of its disruption on lung physiology and pathophysiology at low environmental and dietary levels. In the present study lung tissues from mice exposed to low dose Cd by drinking water (3.3 mg/L, 16 weeks), compared to Cd-free drinking, were examined for redox proteomics (n=3 per group). Redox proteomics was performed by a mass spectrometry-based Isotope Coded Affinity Tag (ICAT) method to measure oxidation of cysteine residues, and pathway and network analyses were performed using MetaCore software. Results showed that 545 protein peptides of two groups were identified and redox states of all 545 were measured (mean % oxidation, control, 32.3%; Cd, 36.4%). Of these, 173 were oxidized 1.3 fold or more by Cd compared to respective protein peptides in control. The results of subcellular compartmental analysis of these 173 showed that mitochondrial proteins were relatively more susceptible to oxidation (1.8 fold) by Cd than proteins in other compartments. Pathway analysis showed that pathways for cystic fibrosis, lung cancer and asthma were associated with the 173 proteins oxidized by Cd, suggesting that exposure to environmental and diet levels of Cd could alter lung redox proteome and impact pulmonary health and diseases.

doi: 10.1016/j.freeradbiomed.2016.10.057

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Nitric Oxide Detection with a Liposome-Encapsulated Spin Trap Donald J. Hirsh1, Brittany M. Schieler2, Katherine M. Fomchenko1, Ethan T. Jordan3, and Kay D. Bidle2 1 The College of New Jersey, Ewing Township, USA, 2Rutgers University, New Brunswick, USA, 3Roger Williams University, Bristol, USA

doi: 10.1016/j.freeradbiomed.2016.10.058 18 Characterisation of the Novel Products of Protein Oxidation by the Inflammatory Oxidant Hypochlorous Acid (HOCl) Using Raman Spectroscopy Maryam Karimi1,2, Michael J. Davies1,2,3, and David I. Pattison1,2 1 The Heart Research Institute, Sydney, Australia, 2Faculty of Medicine, University of Sydney, Australia, 3Panum Institute, University of Copenhagen, Denmark Background: The heme enzyme myeloperoxidase that is released by activated leukocytes at sites of inflammation, uses H2O2 and chloride ions to form the bactericidal agent hypochlorous acid (HOCl). Inappropriate or excessive production of this powerful oxidant can however cause host tissue damage, with this being implicated in multiple human inflammatory diseases. Proteins are major biological targets for HOCl, with sulfur- and amine-containing residues particularly susceptible to damage. Reaction of HOCl with amines (e.g. the -amino groups of oxidised glutathione, GSSG, or the amine group of Lys side-chains) generates chloramines (RNHCl) that undergo subsequent decomposition to aldehydes (R’CHO) and nitriles (R’C≡N). Hypothesis: Nitriles may be characteristic biomarkers of HOCl and chloramine generation, which can be readily detected by Raman spectroscopy via their characteristic absorbance bands. Methods: Sulfur- and aminecontaining model peptides were incubated for 24 h with up to 20fold molar excess HOCl, before drying and analysing by Raman spectroscopy Results: Analysis of the HOCl-treated model peptides by Raman spectroscopy shows dose-dependent changes in the wavelength and intensity of characteristic Raman bands assigned to disulfides (500–550 cm-1), and the formation of a new Raman absorption at ~2255 cm-1 assigned to stretches of nitrile groups. The latter peak was detected in a dose-dependent manner on HOCl treatment of Lys-containing protein and peptide models, N-acetyl-histidine and oxidised glutathione (GSSG). Conclusions: HOCl-mediated oxidation of proteins and peptides

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modifies protein and peptide side-chains in a manner that can be readily detected by Raman spectroscopy. The absorption band attributed to C≡N stretching at ~2255 cm -1 is well removed from other features in the Raman spectra, making it a potential marker of HOCl-mediated protein oxidation, chloramine formation and decomposition.

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doi: 10.1016/j.freeradbiomed.2016.10.059

Vitaly K. Koltover1, Raisa D. Labyntseva 2, Vasilii K. Karandashev 3, and Sergiy O. Kosterin 2 1 Institute of Problems of Chemical Physics, RAS, Russian Federation, 2Palladin Institute of Biochemistry, NAS, Kiev, Ukraine, 3 Institute of Microelectronics Technology and High Purity Materials, RAS, Chernogolovka, Russian Federation

19 New Insights on Effects of a Dietary Supplement Utilizing the "Redox Stress Hypothesis" Daniel Koenig1, Bruno Fink2, and Boris Nemzer3 Institute of Sportmedicine, University of Freiburg, Germany, 2 Noxygen GmbH, Elzach, Germany, 3Future Ceutical Inc., Momence, USA 1

The research community is generally agreed that maintenance of healthy levels of free radicals and related oxidants are important for good health. However, utilization of the “redox stress hypothesis” can provide us with concrete nutritional targets in order to better support and maintain “optimal health.” Following this hypothesis, we performed a single-dose study on the effects of dietary supplement Spectra5TM on oxidative and nitrosative stress markers as well as metabolic, inflammatory and cardiovascular parameters in human participants (n=8). The measurements of ex vivo intra- and extracellular formation of reactive oxygen species (ROS, O2–, H2O2, OH) in whole blood, respiratory activity of blood cells, as well as mitochondrialdependent ROS formation, and respiratory activity were performed using EPR spectrometer NOXYSCAN, spin probe CMH, and oxygen label NOX-15.1, respectively. Furthermore, we investigated the ability of supplement to modulate ex vivo cellular inflammatory responses induced by stimulation with exogenous TNF-α and also followed changes in bioavailable NO concentrations. In this study, we demonstrated that administration of single dose Spectra5TM resulted in statistically significant long-term, time depended inhibition of mitochondrial and cellular ROS generation by as much as 13% as well as 2.5-times inhibition in extracellular NADPH system-dependent generation of O2–, and nearly complete inhibition of extracellular H2O2 formation. This was reflected in more significant inhibition of ex-vivo cellular inflammatory response and also increases in bioavailable NO concentration. In this study we have measured synergetic, biological effects of a natural supplement on changes in oxidative and nitrosative stress as well as cellular metabolic activity. The unique design and activity of the plant-based natural supplement, in combination with the validated “Extended Vitality Test”, demonstrates the efficiency to use the dietary supplements by utilizing the “redox stress hypothesis”.

doi: 10.1016/j.freeradbiomed.2016.10.060

Magnetic-Isotope Effects in ATP Hydrolysis Driven by Myosin as Evidence for a Free-Radical Intermediate in the Reaction

Some chemical elements have magnetic and nonmagnetic stable isotopes. The question arises if the magnetic fields of the atomic nuclei impact on efficiency of biocatalysts. Magnesium cation, Mg2+, serves as cofactor of ATP hydrolysis catalyzed by muscle myosin. Among three stable isotopes, 24Mg, 25Mg, 26Mg with natural abundance 76.7, 10.13, 11.17 %, only 25Mg is magnetic (nuclear spin I = 5/2) while 24Mg and 26Mg are nonmagnetic (I = 0). The effects of different isotopes of Mg on ATPase activity of isolated myosin were studied. It was found that the rate of the enzymatic ATP hydrolysis with 25Mg is 2.0–2.5 times higher as compared to the nonmagnetic 24Mg or 26Mg. The magnetic isotope effect (MIE) was observed at physiological concentrations, 5 mM, of MgCl2. The similar MIE was found with zinc. Among five stable isotopes, 64Zn, 66 Zn, 67Zn, 68Zn, and 70Zn with natural abundance 48.6, 27.9, 4.1, 18.8, and 0.6 %, only 67Zn is magnetic (I = 5/2). While Zn2+ performs the cofactor function less efficiently than Mg2+, it was found that the rate of the enzymatic ATP hydrolysis with 67Zn is 40-50 % higher as compared to the nonmagnetic 64Zn or 68Zn. On its own, MIE unambiguously testifies presence of a spin-selective rate-limiting step as the “bottle-neck” that is accelerated by the magnetic field of nuclear spin of 25Mg or 67Zn. In free-radical chemistry, MIE is the sufficient demonstration of a free-radical or radical ion pair as intermediate in the reaction under study (Buchachenko, 2013). One can assume that, under the condition of the electronconformational excitation, there is a transfer of electron density from NH2 group of Glu459 or OH– group of the bound water molecule to ADP3– or Mg2+/ Zn2+ in the active site of the enzyme with formation of the corresponding radical ion pair. Although detailed mechanisms of ability of myosin to perceive the nuclear magnetism require further investigations, the nuclear spin catalysis opens novel ways in free-radical biomedicine based on the stable magnetic isotopes. Supported by RFBR, project no. 14-04-00593.

doi: 10.1016/j.freeradbiomed.2016.10.061 21 Arginase Inhibitor Reacts with Hemoglobin to Form Nitrite Xiaohua Liu1, Swati Basu1, and Daniel Kim-Shapiro1 1 Wake Forest University, Winston-Salem, USA Endothelial nitric oxide synthase (eNOS) is found in endothelial cells and has recently been found to also be present in red blood cells (RBCs). eNOS can catalyze L-arginine conversion to citrulline and produce nitric oxide (NO). L-arginine can also be converted to L-ornithine and urea through the activity of arginase which consumes L-arginine in blood. Due to their having a common substrate (arginine), arginase would compete with eNOS and limit NO signaling. Inside the RBC, NO formed from NOS is expected to

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