EPR imaging and HPLC characterization of the pigment-based organic free radical in black soybean seeds

We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9?GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn^2+?species based on the g values and hyperfine structures. The signal from the stable radical was noted at g?≈?2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product.     

Spectroscopic characterization of a Ni-organic radical intermediate in the aerobic oxidation of methanol catalyzed by a Ni(II)(polyoximate) complex

In the aerobic oxidation of methanol catalyzed by a Ni(II)(TRISOX) complex [H₃TRISOX = tris(1-propan-2-onyl oxime)amine], an intermediate is observed spectroscopically. The intensities of both the UV-Vis absorption and electron paramagnetic resonance (EPR) spectra associated with this intermediate maximize during the time period of maximum formaldehyde production, and decrease as the methanol oxidation activity decreases. The UV-Vis spectrum has prominent features at 350, 420, and 535 nm. The EPR spectrum is centered at g = 2.00 and shows splittings of 28 ± 5 G. Both of these spectra are consistent with characterization of the intermediate as including one or more iminoxyl radicals derived from the oximate groups of the TRISOX ligand. Spectroscopic features very similar to those in the air-oxidized intermediate are observed in electrochemically oxidized samples, suggesting that the electrochemically generated complex will be a useful model for the intermediate observed during catalytic turnover. The crystal structure of a Ni(II) complex with an intermediate protonation state of the ligand, [Ni(II)₂(H₂TRISOX)₂(μ₂:η¹-ONO₂)](NO₃) · (CH₃CN) · 5(H₂O), 4, has been structurally characterized. Comparison to the previously reported [Ni(II)(H₂TRISOX)(CH₃CN)]₂(ClO₄)₂, 3, shows that bis(μ-oximate) dimers can form either with or without an additional bridging ligand. Addition of the nitrato bridge decreases the Ni-Ni distance from 3.5752(13) Å in 3 to 3.2014(4) Å in 4. It is intriguing to note that the reactions catalyzed by the Ni(II)(TRISOX) complex, the net transfer of two hydrogen atoms from an alcohol or amine substrate to O₂, are the same reactions catalyzed by several different metalloenzymes that also incorporate both a redox active metal and a redox active organic component in their active sites.     

The iron complex of Dp44mT is redox-active and induces hydroxyl radical formation: An EPR study

Iron chelation therapy was initially designed to alleviate the toxic effects of excess iron evident in iron-overload diseases. However, some iron chelator-metal complexes have also gained interest due to their high redox activity and toxicological properties that have potential for cancer chemotherapy. This communication addresses the conflicting results published recently on the ability of the iron chelator, Dp44mT, to induce hydroxyl radical formation upon complexation with iron (B.B. Hasinoff and D. Patel, J Inorg. Biochem.103 (2009), 1093-1101). This previous study used EPR spin-trapping to show that Dp44mT-iron complexes were not able to generate hydroxyl radicals. Here, we demonstrate the opposite by using the same technique under very similar conditions to show the Dp44mT-iron complex is indeed redox-active and induces hydroxyl radical formation. This was studied directly in an iron(II)/H₂O₂ reaction system or using a reducing iron(III)/ascorbate system implementing several different buffers at pH 7.4. The demonstration by EPR that the Dp44mT-iron complex is redox-active confirms our previous studies using cyclic voltammetry, ascorbate oxidation, benzoate hydroxylation and a plasmid DNA strand-break assay. We discuss the relevance of the redox activity to the biological effects of Dp44mT.     

Doxorubicin inhibits oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H(2)O(2) system by reacting with ABTS-derived radical

The effect of doxorubicin on oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS*(+)) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS*(+) radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS*(+) reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS*(+) is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation.     

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods.     

Encapsulation of [Mn(bpy)3] cations in 2D [Mn(dca)3] sheet: Synthesis, X-ray structure and EPR study

The reaction of Mn(NO₃)₂ · 4H₂O, 2,2′-bipyridine (bpy) and sodium dicyanamide (dca) in aqueous medium yielded the {[Mn(bpy)₃][Mn(dca)₃]₂}_n (1). The single-crystal X-ray analysis of 1 revealed that the anionic part of the complex, [Mn(dca)₃]⁻, features infinite 2D sheets with a honeycomb-like porous structure having a void space of ca. 12 Å in which [Mn(bpy)₃]²⁺ cations are encapsulated to yield a fascinating molecular assembly. Mn^II ions possess an octahedral geometry both in the anionic and cationic components of complex 1. In the anionic component, each Mn^II ion is bridged by three pairs of dicyanamide anions in an end-to-end fashion with two other Mn^II ions from adjacent [Mn(dca)₃]⁻ moieties. This type of linking propagates parallel to the bc crystallographic plane to form 2D sheets. [Mn(bpy)₃]²⁺ is found to have somewhat “squeezed” upon encapsulation. No measurable magnetic interaction was evidenced through variable temperature magnetic susceptibility measurements. However, in addition to the broad g ≈ 2 resonance typical of magnetically diluted [Mn(bpy)₃]²⁺ cations, EPR spectroscopy evidenced exchange narrowing of the [Mn(dca)₃]⁻ resonance at g ≈ 2 thus indicating operation of weak magnetic interactions extended over the whole 2D network through the dca⁻ bridges.     

Optical and EPR spectra of the thionitrosyl complex [Cr(OH2)5(NS)]

The green thionitrosyl complex [Cr(OH₂)₅(NS)]²⁺ was isolated in solution by the hydrolysis of [Cr(NCCH₃)₅(NS)]²⁺. The optical absorption spectra of both compounds are dominated by a band with vibrational progression around 600 nm assigned as a {d_yz,zx, π^∗(NS)} → {π^∗(NS), d_yz,zx}^∗ transition. The optical data indicate that the NS ligand is a weaker π-acceptor than the NO ligand. The EPR parameters of [Cr(OH₂)₅(NS)]²⁺ were determined: g_iso, g_∥ and g_⊥: 1.96515, 1.92686(5) and 1.986860(8); A_iso(⁵³Cr), A_∥(⁵³Cr) and A_⊥(⁵³Cr): 25.3 × 10⁻⁴, 38 × 10⁻⁴ and 18.5 × 10⁻⁴ cm⁻¹; A_iso(¹⁴N), A_∥(¹⁴N) and A_⊥(¹⁴N): 6.5 × 10⁻⁴, 2.81 × 10⁻⁴ and 8.346(12) × 10⁻⁴ cm⁻¹.     

EPR study of the oxidation reaction of nickel(0) phosphine complexes with Lewis and Brønsted acids

The oxidation of Ni(PPh₃)₄ with BF₃ · OEt₂, H₃CCOOH, and F₃CCOOH, and that of (PPh₃)₂Ni(C₂H₄) with BF₃ · OEt₂ is studied by EPR spectroscopy. The reaction of the Ni(0) complexes with BF₃ · OEt₂ gives Ni(II) complexes with which they react to form Ni(I) compounds with covalent Ni-F and Ni-B bonds that transform with excess BF₃ · OEt₂ into cationic paramagnetic Ni(I) complexes. Acetic acid also adds oxidatively to Ni(PPh₃)₄ to form a Ni(II) complex that reacts further to give Ni(I) hydride and carboxylate complexes. The Ni(I) hydride is transformed by the acid into the Ni(I) carboxylate with release of hydrogen, the amount of which depends on the rate of acid addition. The following Ni(I) complexes are identified in the reaction medium: [Ni(PPh₃)₃]BF₄, [(PPh₃)₂Ni(OEt₂)]BF₄, [(PPh₃)Ni(OEt₂)_n]BF₄, (PPh₃)₂NiBF₂, (PPh₃)₃NiOOCCH₃, and [(PPh₃)₂Ni(OEt₂)P(OEt)₃]BF₄. Oxidation schemes of Ni(0) complexes by Lewis and Brønsted acids are given.     

The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.     

Kinetic simulation studies on the transient formation of the oxo‐iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)‐porphyrin with hydrogen peroxide in aqueous solution

High‐valent oxo‐iron(IV) species are commonly proposed as the key intermediates in the catalytic mechanisms of iron enzymes. Water‐soluble iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) has been used as a model of heme‐enzyme to catalyse the hydrogen peroxide (H₂O₂) oxidation of various organic compounds. However, the mechanism of the reaction of Fe(III)TMPyP with H₂O₂ has not been fully established. In this study, we have explored the kinetic simulation of the reaction of Fe(III)TMPyP with H₂O₂ and of the catalytic reactivity of FeTMPyP in the luminescent peroxidation of luminol. According to the mechanism that has been established in this work, Fe(III)TMPyP is oxidized by H₂O₂ to produce (TMPyP)^·+Fe(IV)=O (k1 = 4.5 × 10⁴/mol/L/s) as a precursor of TMPyPFe(IV)=O. The intermediate, (TMPyP)^·+Fe(IV)=O, represented nearly 2% of Fe(III)TMPyP but it does not accumulate in suf?cient concentration to be detected because its decay rate is too fast. Kinetic simulations showed that the proposed scheme is capable of reproducing the observed time courses of FeTMPyP in various oxidation states and the decay pro?les of the luminol chemiluminescence. It also shows that (TMPyP)^·+Fe(IV)=O is 100 times more reactive than TMPyPFe(IV)=O in most of the reactions. These two species are responsible for the initial sharp and the sustained luminol emissions, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

Rigid core and flexible terminus: structure of solubilized light-harvesting chlorophyll a/b complex (LHCII) measured by EPR

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations.     

Involvement of malate,monophenols, and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Peroxidase associated with isolated horseradish cell walls catalyzes the formation of H₂O₂ in the presence of NADH. The reaction is stimulated by various monophenols, especially of coniferyl alcohol. NADH can be provided by a bound malate dehydrogenase. This system is capable of polymerizing coniferyl alcohol yielding an insoluble dehydrogenation polymer. NADH was found to be oxidized by two different mechanisms, one involving Mn²⁺, monophenol, and the superoxide radical O₂ ^·- in a reaction that is not affected by superoxide dismutase, and another one depending on the presence of free O₂ ^·- and probably of an enzyme-NADH complex. A scheme of these reaction chains, which are thought to be involved in the lignification process, is presented.Abbreviations DHP dehydrogenation polymer - GOT glutamate oxaloacetate transaminase (EC 2.6.1.1) - LDH lactate dehydrogenase (pig heart, EC 1.1.1.27) - MDH malate dehydrogenase (EC 1.1.1.37) - pCA p-coumaric acid - SOD superoxide dismutase (EC 1.15.1.1) - TLC thin-layer chromatography - XOD xanthine oxidase (EC 1.2.3.2)     

Synthesis and characterization of [Co(NO2)2(acac)(IMH2py)] with one-electron-reduced imino nitroxide radical associated with unusual displacement of acetylacetonate in the starting complex trans-Na[Co(NO2)2(acac)2]

A reaction of trans-Na[Co(NO₂)₂(acac)₂] with IM2py(2(2^′-pyridyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-1-oxyl) in methanol afforded trans-[Co(NO₂)₂(acac)(IMH2py)](IMH2py=1-hydroxyl-2(2^′-pyridyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole); one-electron reduction of the N-O radical moiety in IM2py and displacement of one of the two acac ligands with retention of two nitrito ligands in the starting complex during the reaction. This new complex was characterized by UV-Vis, ¹H NMR spectra and X-ray analysis.     

Study of the O-Ru-N bonding in trans-[Ru(NH3)4(SO4)L] complexes (L=imidazole, histidine and substituted pyridines): a X-ray, EPR, spectroscopic and theoretical MO study

Spectroscopic studies on trans-[Ru(NH₃)₄(SO₄)L]⁺ where L=imidazole, histidine, pyridine and substituted pyridines were undertaken to understand the effect of various ligands on the Ru-N bonding in these complexes. The sulfate complexes show two major bands in the 250-270 and 310-350 nm region of the UV-Vis spectrum. Based on quantum chemical calculations the lowest energy band has been assigned to a LMCT (SO₄ ²⁻ → Ru^III) transition. The energy of the LMCT transition decreases as the order of the axial ligand L basicity: Him > L-hist > 4-NH2-py > 4-Cl-py > 4-pic > py > nia > 4-Cn-py > isn > pz. EPR spectra give only two g values showing that the two LUMO containing the metal d_π orbitals are degenerate and the energy separation between the LUMO and HOMO, calculated from the g values correlates linearly with the charge transfer energy and electrochemical properties. These correlations suggest extensive π donation from L to the Ru(III) d orbitals. An X-ray study of the 4-pic complex shows a bent S-O-Ru bond of 127.5° and MO calculations for three other complexes predict similar angles due to extensive σ and π bonding interaction between the sulfate oxygen and the Ru(III) ion. Surprisingly, the MO calculations do not predict the observed degeneracy in the LUMO orbital found by EPR studies. We shall argue that these discrepancies can be reconciled by insisting that the orientation of the L ring be coplanar with the S-O-Ru plane as is the case in the one X-ray study.     

Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): a combined experimental and theoretical study

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.     

EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ

The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron–sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q₁) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQ_Nf (fast-relaxing component), SQ_Ns (slow-relaxing), and SQ_Nx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQ_Nf, because of its high sensitivities to and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQ_Nf signal at a much lower concentration than that required to quench the slower relaxing components (SQ_Ns and SQ_Nx). A close correlation was shown between the line shape alteration of the g = 2.05 signal of the cluster N2 and the quenching of the SQ_Nf signal, using two different experimental approaches: (1) changing the poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQ_Nf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I.     

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes

Background

Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential.

Conclusions

Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function.

Methods

To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration.

Results

AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10–30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration.

General significance

These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

Spectrophotometric evidence for the formation of short-lived hypomanganate(V) and manganate(VI) transient species during the oxidation of K-carrageenan by alkaline permanganate

Spectrophotometric detection of the formation of short-lived hypomanganate(V), [KCAR-Mn(V)O43-], and manganate(VI), [KCAR-Mn(VI)O42-], intermediates has been confirmed through the oxidation of K-carrageenan (KCAR) by potassium permanganate in alkaline solutions of pH's >or= 12 using a conventional spectrophotometer. The short-lived transient species were characterized and a mechanism consistent with experimental observations is suggested.