Electron Paramagnetic Resonance Spin Trapping Research Articles

ESR spin trapping determination of the hydroperoxide concentration in polyethylene oxide (PEO) in aqueous solution

Initial hydroperoxide (ROOH), which exists even in freshly produced polymers, acts as an initiator for the oxidative degradation of the polymer. Electron spin resonance (ESR) with the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used to investigate the presence of ‘initial ROOH’ in a polyethylene oxide (PEO) sample. The degradation of PEO was investigated by heating or irradiating mixtures of PEO/DMPO/H2O with or without dissolved oxygen by ultraviolet-visible (UV-vis, λ > 250 nm) and visible (λ > 350 nm) light. The concentration of ‘initial ROOH’ and the photolysis and thermolysis rate constants were estimated from the concentrations of the DMPO–H, DMPO–C–PEO, and DMPO–OH spin adducts. Knowledge of the oxidative degradation mechanism of polymers allows the ‘initial ROOH’ concentration to be used as a quality control indicator for polymers.

Generation of the SCF3 Radical by Photoredox Catalysis: Intra- and Intermolecular Carbotrifluoromethylthiolation of Alkenes.

We report the first use of N-trifluoromethylthiosaccharin as the source of SCF3 radical under photoredox catalysis. This allowed an efficient and general visible-light-mediated carbotrifluoromethylthiolation of alkenes. Under the optimized conditions using fac-[Ir(ppy)3 ] as the photocatalyst, various N-aryl acrylamides as well as a wide range of substituted styrenes can readily be difunctionalized in an intra- or intermolecular fashion, affording the corresponding SCF3 -containing products in good to excellent yield. Importantly, the formation of this SCF3 radical along with other key radical intermediates was unambiguously demonstrated thanks to spin trapping/electron paramagnetic resonance (ST/EPR) experiments, which enabled a clear understanding of the reaction mechanism.

Kinetics and Mechanism of Ultrasonic Activation of Persulfate: An in Situ EPR Spin Trapping Study.

Ultrasound (US) was shown to activate persulfate (PS) providing an alternative activation method to base or heat as an in situ chemical oxidation (ISCO) method. The kinetics and mechanism of ultrasonic activation of PS were examined in aqueous solution using an in situ electron paramagnetic resonance (EPR) spin trapping technique and radical trapping with probe compounds. Using the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radical (•OH) and sulfate radical anion (SO4•-) were measured from ultrasonic activation of persulfate (US-PS). The yield of •OH was up to 1 order of magnitude greater than that of SO4•-. The comparatively high •OH yield was attributed to the hydrolysis of SO4•- in the warm interfacial region of cavitation bubbles formed from US. Using steady-state approximations, the dissociation rate of PS in cavitating bubble systems was determined to be 3 orders of magnitude greater than control experiments without sonication at ambient temperature. From calculations of the interfacial volume surrounding cavitation bubbles and using the Arrhenius equation, an effective mean temperature of 340 K at the bubble-water interface was estimated. Comparative studies using the probe compounds tert-butyl alcohol and nitrobenzene verified the bubble-water interface as the location for PS activation by high temperature with •OH contributing a minor role in activating PS to SO4•-. The mechanisms unveiled in this study provide a basis for optimizing US-PS as an ISCO technology.

Plasmonic Bi nanoparticles and BiOCl sheets as cocatalyst deposited on perovskite-type ZnSn(OH)6 microparticle with facet-oriented polyhedron for improved visible-light-driven photocatalysis

Perovskite-type structure materials have attracted considerable attention in the field of solar cell and photocatalysis. In this study, novel plasmonic Bi nanoparticles and BiOCl sheets co-decorated ZnSn(OH)6 (ZSH) with various morphologies (hence exposure facets) was successfully synthesized via one-pot precipitation, hydrolysis and UV-photoreduction process. The as-obtained materials were characterized by field emission scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, ultraviolet–visible diffuse reflection spectroscopy and photocurrent-time measurement. Results indicated that the cubic, 14-facets polyhedral and octahedral ZSH were decorated by plasmonic Bi particles and BiOCl sheets, which improved visible-light absorption, charge carrier separation and migration performance. Compared with pure ZSH, the composites showed enhanced photocatalytic activity, with the highest photocatalytic performance achieved by the octahedral ZSH decorated by Bi and BiOCl (i.e., ZSH-3-Bi). The photo-degradation rate of ZSH-3-Bi was about 81 times higher than that of pure octahedral ZSH. This phenomenon was mainly ascribed to the surface plasmon resonance effect for visible light absorption, the exposed {111} facets relative to other facets of octahedral ZSH, and the improved charge carrier separation efficiency due to the strong interfacial interaction of three components. The radical and hole trapping, and electron spin resonance spin-trap experiments verified that the OH, O2− and h+ were the main reactive species in this system, which were responsible for the decoloration and part mineralization of the RhB molecules. Therefore, this work provides new insights into the in situ fabrication of plasmonic Bi-based photocatalyst with facet-oriented polyhedron for full utilization of solar energy and wastewater treatment.

Integrating AgI/AgBr biphasic heterostructures encased by few layer h-BN with enhanced catalytic activity and stability

Using freshly prepared water-soluble KBr crystal as facile, low-cost sacrificial template, AgBr nanocubes were synthesized through one-pot precipitation method, then navy bean shaped AgI/AgBr biphasic heterostructures were synthesized through anion-exchange reaction and encased within few-layer h-BN to obtain final product. The obtained heterostructured AgI/AgBr/h-BN composite without plasmonic noble metal nanoparticles was used as stable and high active photocatalyst for dye degradation under visible light irradiation, comparing both with self-prepared normal AgBr, AgBr cubes, AgI/AgBr navy beans and other related catalysts reported in the literature. The significant boosting of activity was attributed to the formation of AgI/AgBr interface and the coupling of few-layer h-BN, the latter of which not only effectively suppresses the reduction of silver ions but greatly enhance the charge separation. Furthermore, it was suggested that the photogenerated holes and superoxide radical were the main active species according to photoelectron chemical measurements, electron spin resonance spin-trap analysis and radical trapping experiments. Finally, the possible mechanism of enhanced photocatalytic activity and stability was discussed and proposed. The work demonstrates that engineering Ag-based semiconductor coupling with h-BN would profit the design strategy for low-cost, solar-driven photocatalysts.

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1⋅s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

Suplatast tosilate protects the lung against hyperoxic lung injury by scavenging hydroxyl radicals

Prolonged exposure to hyperoxia produces extraordinary amounts of reactive oxygen species (ROS) in the lung and causes hyperoxic lung injury. Although supraphysiological oxygen is routinely administered for the management of respiratory failure, there is no effective strategy to prevent hyperoxic lung injury. In our previous study, we showed that suplatast tosilate, an asthma drug that inhibits T helper 2 (Th2) cytokines, ameliorated bleomycin-induced lung injury and fibrosis through Th2-independent mechanisms. Because bleomycin also generates ROS, we hypothesized that suplatast tosilate might have antioxidant activity and protect the lung against hyperoxic lung injury. To test this hypothesis, mice exposed to hyperoxia were given suplatast tosilate through drinking water. Treatment with suplatast tosilate significantly prolonged mouse survival, reduced the increases in the numbers of inflammatory cells, levels of the pro-inflammatory cytokines/chemokines IL-6 and MCP-1, and protein in bronchoalveolar lavage fluid, and ameliorated lung injury in histological assessment. Suplatast tosilate treatment also significantly inhibited hyperoxia-induced elevations in the levels of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in bronchoalveolar lavage fluid and 8-isoprostane, a marker of lipid peroxidation, in lung tissue. This finding suggests that suplatast tosilate exerts an antioxidant activity in vivo. In addition, we investigated whether suplatast tosilate has a scavenging effect on hydroxyl radical, the most reactive and harmful ROS, using electron paramagnetic resonance spin-trapping. Suplatast tosilate was shown to scavenge hydroxyl radicals in a dose-dependent manner, and its reaction rate constant with hydroxyl radical was calculated as 2.6×1011M−1S−1, which is faster than that of several well-established antioxidants, such as ascorbate, glutathione, and cysteine. These results suggest that suplatast tosilate protects the lung against hyperoxic lung injury by decreasing the degree of oxidative stress induced by ROS, particularly by scavenging hydroxyl radicals. Suplatast tosilate might become a potential therapeutic for hyperoxic lung injury.

Cathodic reductive couplings and hydrogenations of alkenes and alkynes catalyzed by the B12 model complex

The reductive coupling and hydrogenation of alkenes were catalyzed by the B12 model complex, heptamethyl cobyrinate perchlorate (1), in the presence of acid during electrolysis at −0.7 V vs. Ag/AgCl in acetonitrile. Conjugated alkenes showed a good reactivity during electrolysis to form reduced products. The product distributions were dependent on the substituents at the CC bond of the alkenes. ESR spin-trapping experiments using 5,5-dimethylpyrroline N-oxide (DMPO) revealed that the cobalt-hydrogen complex (CoH complex) should be formed during the electrolysis and it functioned as an intermediate for the alkene reduction. The electrolysis was also applied to an alkyne, such as phenylacetylene, to form 2,3-diphenylbutane (racemic and meso) and ethylbenzene via styrene as reductive coupling and hydrogenated products, respectively.

Revisiting the catalytic activity of a doped SrFeO3 for water pollutants removal: Effect of light and temperature

A Sr0.85Ce0.15FeO3-δ perovskite-type mixed oxide was prepared by solution combustion synthesis from citric acid and carefully characterized for the structure by X-ray diffraction coupled with Rietveld analysis, for the microstructure/morphology by HRTEM and SEM, for texture by N2 uptake at low temperature, for surface charge by ζ potential measurements and for reactive species by Electron Paramagnetic Resonance (EPR). Photocatalytic activity was tested in the presence of simulated solar light towards Orange II and Rhodamine B as model pollutants. Despite this compound is known as photocatalyst, a good reactivity was evidenced in the 55–80°C temperature range without light irradiation, indicating that it can work as well in the dark after thermal-activation. Therefore, experimental data obtained in this work suggest to carefully reconsidering the role of this mixed oxide as photocatalyst. EPR spin-trapping tests allowed to deepen the aspects related to reactive species generated by Sr0.85Ce0.15FeO3-δ, revealing that the temperature increase promotes hydroxyl radicals and singlet oxygen production. Furthermore, an interesting selectivity towards the degradation of different dyes was found.

“Dark” Singlet Oxygen and Electron Paramagnetic Resonance Spin Trapping as Convenient Tools to Assess Photolytic Drug Degradation

Forced degradation studies are an important tool for a systematic assessment of decomposition pathways and identification of reactive sites in active pharmaceutical ingredients (APIs). Two methodologies have been combined in order to provide a deeper understanding of singlet oxygen-related degradation pathways of APIs under light irradiation. First, we report that a “dark” singlet oxygen test enables the investigation of drug reactivity toward singlet oxygen independently of photolytic irradiation processes. Second, the photosensitizing properties of the API producing the singlet oxygen was proven and quantified by spin trapping and electron paramagnetic resonance analysis. A combination of these techniques is an interesting addition to the forced degradation portfolio as it can be used for (1) revealing unexpected degradation pathways of APIs due to singlet oxygen, (2) clarifying photolytic drug-drug interactions in fixed-dose combinations, and (3) synthesizing larger quantities of hardly accessible oxidative drug degradants.

Efficient sterilization using reactive oxygen species generated by a radical vapor reactor

Abstract Numerous sterilization techniques using ozone have been proposed. However, singlet oxygen and hydroxyl radicals show stronger oxidation activity than ozone, and therefore exert a greater sterilization effect against microorganisms. We recently developed a radical vapor reactor (RVR) as a powerful tool for microorganism sterilization, which is based on the efficient generation and exposure of reactive oxygen species (ROS) such as singlet oxygen and hydroxyl radicals including ozone. Therefore, effective sterilization via ROS generation is expected by optimizing the RVR conditions. In this study, the generations of ROS was measured under various RVR conditions using spin-trapping electron spin resonance analysis and ozone monitoring. We demonstrated the effective sterilization of Escherichia coli and Bacillus subtilis by exposure to ozone, singlet oxygen, and hydroxyl radicals whose production levels were successfully controlled.

Techniques for Detecting Reactive Oxygen Species in Pulmonary Vasculature Redox Signaling.

Redox signaling plays important roles in regulating pulmonary vasculature function. Aberrant redox signaling, e.g., overproduction of reactive oxygen species (ROS) that exceeds the capability of cellular antioxidant mechanisms, has been found to alter vasculature function and remodel blood vessel structure, thus contributes to pathological processes of pulmonary vasculature. The regulation of pulmonary vasculature via ROS is a very complicated process with various biological events involved, however, the specific effect of individual ROS and the underlying mechanism still remain unclear. Most of ROS are present as free radical forms with extremely short lifetime, which makes it very difficult to detect the ROS and investigate their bioactivities. Therefore, developing specific and sensitive methods to detect ROS in complex biological system is essential for us to advance our knowledge in pulmonary vasculature regulation. In this chapter, we introduce several commonly used techniques for the detection of ROS in vitro and in vivo, including chemiluminescence-based assay, fluorescence-based assay, cytochrome c reduction method, genetically encoded fluorescent probes, as well as ESR spin trapping technique. We also discuss the advantages, limitations, and recent technical advances of each individual technique as well as their applications in pulmonary vasculature studies. We believe that technical advance in the detection of ROS will provide us with a better understanding on how to maintain normal pulmonary vasculature functions under oxidative stress.

In vitro biocompatibility of a ferrimagnetic glass-ceramic for hyperthermia application

Ferrimagnetic glass-ceramics containing magnetite crystals were developed for hyperthermia applications of solid neoplastic tissue. The present work is focused on in vitro evaluation of the biocompatibility of these materials, before and after soaking in a simulated body fluid (SBF). X-ray diffraction, scanning electron microscopy, atomic absorption spectrophotometry, X-ray photoelectron spectrometry and pH measurements were employed in glass-ceramic characterisation. The free-radical mediated reactivity of the glass-ceramic was evaluated by Electron Paramagnetic Resonance (EPR) spin trapping. Cell adhesion and proliferation tests were carried out by using 3T3 murine fibroblasts. Cytotoxicity was performed by qualitative evaluation of human bone osteosarcoma cells U2OS cell line. The results show that almost two times more 3T3 cells proliferated on the samples pre-treated in SBF, compared with the untreated specimens. Moreover a decrease of confluence was observed at 48 and 72h for U2OS cells exposed to the untreated glass-ceramic, while the powder suspensions of glass-ceramic pre-treated in SBF did not influence the cell morphology up to 72h of exposition. The untreated glass-ceramic exhibited Fenton-like reactivity, as well as reactivity towards formate molecule. After pre-treatment with SBF the reactivity towards formate was completely suppressed. The concentration of iron released into the SBF solution was below 0.1ppm at 37°C, during one month of soaking. The different in vitro behaviour of the samples before and after SBF treatment has been correlated to the bioactive glass-ceramic surface modifications as detected by morphological, structural and compositional analyses.

Evaluation of Methanol Induced Free Radicals in Mice Liver

Methanol induced oxidative stress (OS) models in mice were successfully established and evaluated by the electron paramagnetic resonance (EPR) spin trapping technique. The capacity for removal of reactive oxygen species (ROS) free radicals by rhubarb and vitamin C (Vc) as candidate materials was also investigated. EPR was employed to determine the free radicals generated from a spin trapping agent, α-phenyl-N-tert-butylnitrone (PBN), that reacted with the ROS. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and the level of malondialdehyde (MDA) were also evaluated by enzyme assays. The results indicated that methanol clearly promoted the generation of ROS free radicals in the liver of mice. The activity of SOD and GSH-PX was reduced significantly, although the level of MDA was increased as a result of the harmful effect of methanol. In addition, rhubarb and Vc exhibited a protective effect on the mice liver under acute OS.

DFT calculations as a powerful tool for ESR spin trapping experiments

A combined electron spin resonance (ESR) and density functional theory study has been carried out to obtain deeper insight in the reaction mechanism of the photorearrangement of 3-hydroxyindolic nitrones. Also, in this case, the combination of these techniques constitutes a powerful tool when discriminating between different reaction pathways. In particular, density functional theory calculations played an important role for unraveling the overlapping of signals coming from different species in the ESR spin trapping experiments. In the present study, the computed energies and the corresponding ESR parameters of each possible isomer hypothesized have been considered, taking also into account their different possibilities of intramolecular H-bond formation. However, the results obtained indicate that the possibility of intramolecular H-bonding did not play a determinant role in this case. Copyright © 2016 John Wiley & Sons, Ltd.

Molecular Structure of Poly(vinyl alcohol)-Derived Carbon-Centered Radicals Studied by Rapid-Flow and Spin-Trapping ESR Measurements: A Short-Lived Intermediate Radical in the Initial Stage of the Graft Polymerization Reaction

Abstract Continuous rapid-flow electron spin resonance (ESR) was used to detect poly(vinyl alcohol) (PVA)-derived carbon-centered radicals, in order to characterize the short-lived intermediate radicals in the initial stage of the graft polymerization of PVA. ESR spectra were recorded by mixing PVA, hydrogen peroxide, and Ti2(SO4)3 at pH 2.0. The observed spectrum indicated three paramagnetic species: a minor species A, and two major species B1, and B2. Based on the observed proton hyperfine coupling constant (hfcc) values, species A was assigned to the carbon-centered radical of PVA produced by hydrogen atom abstraction from the methine carbon bearing a hydroxyl group. Species B1 and B2 were ascribed to two structural isomers of the PVA-derived, carbonyl-conjugated radical produced by acid-catalyzed radical reaction at the vicinal diol moiety (i.e., head-to-head structure) of PVA. Spin-trapping ESR measurements were performed at 80 °C using a water-soluble spin-trapping reagent 3,5-dibromo-4-nitroso-benzenesulfonate sodium salt (DBNBS). Based on the hfcc values observed for the spin-adducts, species B1 and B2 were confirmed to be the major PVA main-chain-derived radicals produced under the graft reaction conditions. These findings demonstrated that the PVA-derived, carbonyl-conjugated radicals are one of the intermediate radicals in the initial stage of the graft polymerization reaction under acidic conditions.

207 - Redox Activity Induced by Metal Coordination with Prion Peptide

Background Physiological function of prion protein (PrP) is still unknown, however its redox and metal-reservoir potentials are viewed as meaningful. We also showed the conformational change of the human PrP binding to Cu2+ [1]. In this report, we have examined the redox ability and the stability of metals at binding site using octarepeat peptide, which is a primary site for Cu2+ uptake in the N-terminal region of PrP. Materials and Methods An eight-residue peptide (PHGGGWGQ) of prion protein was used for the spectroscopic affinity assay for metal ions (Cu2+, Ni2+, Co2+, Zn2+, and Mn2+). For electron spin resonance (ESR), the peptide and Cu2+ were prepared as 0.1 mM in 10 mM HEPES buffer (pH 7.6) containing 30% glycerol. The ESR spectrum was acquired at 20 K. For circular dichroism (CD), the peptide and Cu2+ were prepared as 0.8 mM in 15 mM HEPES buffer. To verify competitive metal binding, octapeptide-Cu2+ was titrated with other divalent metal ions such as Ni2+, Co2+, Zn2+, and Mn2+. The reactivity of metal-containing peptide with ROS was analyzed by ESR spin trapping with CYPMPO. Results The ESR at 20K showed two types of coordination modes for the binding of octapeptide-Cu2+. The CD spectrum of octapepide-Cu2+ indicated the characteristic positive peak of molecular ellipticity at visible wavelengths, which was also assigned to Cu2+ coordination complexes. The CD peak of octapeptide-Cu2+was reduced by titrating with other divalent metal ions, such as Ni2+, Co2+, Zn2+, and Mn2+. ESR spin trapping showed that the copper-peptide complex converted superoxide into hydroxyl radical.

95 - Fenamic Acid Analogues Oxidized by Peroxidase Enzymes Cause the Formation of Fibronectin Aggregation and Fibronectin-Derived Radicals

Fenamic acids form the substructure of many non-steroidal anti-inflammatory drugs (NSAIDs); they are amongst the most commonly prescribed drugs globally. Interactions between fenamic acid NSAIDs and peroxidases have shown that reactive metabolites can be produced, although detailed studies of these metabolites or their relative reactivity have not been studied. Adverse drug reactions may be catalyzed by peroxidase enzymes in off-target tissues. Myeloperoxidase (MPO) is a peroxidase enzyme found abundantly in neutrophils, which are recruited during inflammation and it is well known that MPO can catalyze drug oxidation. Moreover, it has been demonstrated that MPO binds to fibronectin (FN); the latter is an important multi-functional protein. The objective of this study was to examine the differential reactivity of fenamic acid NSAIDs when oxidized by MPO and the consequent effects on FN. Studies using ESR spin trapping with DMPO revealed that N-phenanthranilic acid (N-PA) produced a stable radical when oxidized by MPO/H2O2; diphenylamine (DPA) and 3-methyldiphenylamine (3-MDA) produced ●OH (DMPO/●OH). In addition, the analogs showed the formation of GS● after the addition of GSH to the MPO/H2O2 system, with 3-MDA producing the most intense DMPO/GS● spectrum. To determine the cytotoxic potential of these radical metabolites, we used HL-60 promyelocytic leukemia cells which contain MPO. 3-MDA, DPA and N-PA demonstrated MPO-catalyzed cytotoxicity in these cells, but not mefenamic acid (MFA). LC/MS analysis of the DPA product formation showed the formation of a hydroxydiphenylamine and a quinoneimine product, indicating MPO oxidation resulted in a hydroxylation reaction. The quinoneimine products appeared to react with GSH, forming a GSH conjugate. Immuno-spin trapping analysis demonstrated that MFA was the most potent in producing FN-protein free radical formation in a MPO-system. However, FN aggregation analysis by silver staining was most prominent with DPA. MFA and its analogues showed the formation of free radicals in a peroxidase-system which interferes with normal cell functioning. Future studies are needed to determine the functional impact of fenamic acid NSAID mediated damage to FN.

401 - A Radical Pathway for Minocycline Toxicity: Role of Cytochrome c and Sulfite

Minocycline (MC) is a second-generation semi-synthetic analogue of tetracycline. MC has been used as an antibiotic and for the treatment of various diseases such as acne vulgaris, sexually transmitted diseases, and rheumatoid arthritis. MC is currently being explored in various clinical trials for the treatment of cardiovascular diseases such as stroke, vascular aneurisms, and coronary by-pass surgery. However, the clinical use of MC has been associated with potential side effects. It has been demonstrated that MC interacts with cytochrome c (Cyt-c) and inhibits its peroxidase activity. However, molecular mechanism involved in the interaction between Cyt-c and MC is not studied. In this study, UV-visible spectroscopic measurements were carried out to understand the reaction between ferric cytochrome c (Fe3+Cyt-c) and MC. MC reduces Fe3+Cyt-c to ferrous cytochrome c (Fe2+Cyt-c). The reduction of Fe3+Cyt-c increases with increasing concentration of MC. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with Fe3+Cyt-c, MC, and sulfite. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3–) was obtained. The formation of DMPO-SO3– was very low in the absence of MC. Interestingly, the addition of MC enhanced the DMPO-SO3– adducts. Furthermore, the formation of DMPO-SO3– adducts increased with increasing concentration of MC. From these results, we propose a mechanism that the Fe3+Cyt-c oxidizes MC to MC radical, which subsequently oxidizes sulfite to sulfite radical. Our results suggest that Fe3+Cyt-c could have a novel role in the deleterious effects of MC in biological systems by increasing the production of sulfite radical.

248 - Catalysis of Sulfite Oxidation Adds to the Array of Biologically Relevant Reactions of Cationic Mn(III) Porphyrins, Widely Known as SOD-Mimics

Sulfite is generated in vivo as a result of metabolism of sulfur containing amino acids methionine and cysteine. Sulfite is consumed as antioxidant/preservative in food, beverages and medications. We are also consuming it through exposure to major environmental pollutant - sulfur dioxide. The enzyme sulfite oxidase (SO) is responsible for the detoxification of sulfite to sulfate, which is subsequently excreted into urine. The SO deficiency is one of the most common causes of sulfite hypersensitivity and toxicity. Herein we report for the first time the ability of cationic Mn(III) N-substituted pyridyl(imidazolyl)-porphyrins (MnPs) to catalyze sulfite oxidation. Originally developed as SOD mimics, MnPs operate at Mn III P/Mn II P reduction potential that is highly compatible with reduction potentials of numerous biomolecules and reactive species, among them superoxide, peroxynitrite, oxygen, hydrogen peroxide, hypochlorite, nitric oxide, thiols, and ascorbate. At least 4 oxidation states of Mn in MnPs (+2, +3, +4 and +5) are involved in their actions which may occur via one- or two-electron transfers. We are here providing evidence, based on spectrophotometric measurements, that several analogs of MnP are able to catalyze sulfite oxidation one-electronically via employing Mn III P/Mn II P redox couple. The reaction proceeds through generation of sulfite radical anion as an intermediate species characterized by electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Clark electrode measurements of the MnP/sulfite system indicated that molecular oxygen is also involved, both in regeneration of Mn III P and further oxidation of sulfite radical to sulfate. Such redox further substantiates tight connections of MnP bioeffects to sulfur biology and indicates that we are still in infancy with regards to fully understanding the plethora of reactions of redox-active drugs. The MnP/sulfite cycling is the reminiscence of redox behavior of MnP/ascorbate system, which has been used successfully in tumor models, suggesting its therapeutic potential. Support: NC Biotechnology# 2016-BIG-6518