Oxidation of di- and tripeptides of tyrosine and valine mediated by singlet molecular oxygen, phosphate radicals and sulfate radicals.

Kinetics and mechanism of the oxidation of tyrosine (Tyr) and valine (Val) di- and tripeptides (Tyr-Val, Val-Tyr and Val-Tyr-Val) mediated by singlet molecular oxygen [O(2)((1)Delta(g))], phosphate (HPO(4)(*-) and PO(4)(*2-)) and sulfate (SO(4)(*-)) radicals was studied, employing time-resolved O(2)((1)Delta(g)) phosphorescence detection, polarographic determination of dissolved oxygen and flash photolysis. All the substrates were highly photooxidizable through a O(2)((1)Delta(g))-mediated mechanism. Calculated quotients between the overall and reactive rate constants for the quenching of O(2)((1)Delta(g)) by Tyr-derivatives (k(t)/k(r) values, accounting for the efficiency of the effective photooxidation) were 1.3 for Tyr, 1 for Tyr-Val, 2.8 for Val-Tyr and 1.5 for Val-Tyr-Val. The effect of pH on the kinetics of the photooxidative process confirms that the presence of the dissociated phenolate group of Tyr clearly dominates the O(2)((1)Delta(g)) quenching process. Products analysis by LC-MS indicates that the photooxidation of Tyr di- and tripeptides proceeds with the breakage of peptide bonds. The information obtained from the evolution of primary amino groups upon photosensitized irradiation is in concordance with these results. Absolute rate constants for the reactions of phosphate radicals (HPO(4)(*-) and PO(4)(*2-), generated by photolysis of the P(2)O(8)(4-) at different pH) and sulfate radicals (SO(4)(*-), produced by photolysis of the S(2)O(8)(2-)) with Tyr peptides indicate that for all the substrates, the observed tendency in the rate constants is: SO(4)(*-) > or = HPO(4)(*-) > or = PO(4)(*2-). Formation of the phenoxyl radical of tyrosine was detected as an intermediate involved in the oxidation of tyrosine by HPO(4)(*-).     

Disproportionation pathways of aqueous hyponitrite radicals (HN2O2(*)/N2O2(*-))

Pulse radiolysis and flash photolysis are used to generate the hyponitrite radicals (HN2O2(*)/N2O2(*-)) by one-electron oxidation of the hyponitrite in aqueous solution. Although the radical decay conforms to simple second-order kinetics, its mechanism is complex, comprising a short chain of NO release-consumption steps. In the first, rate-determining step, two N2O2(*-) radicals disproportionate with the rate constant 2k = (8.2 +/- 0.5) x 10(7) M(-1) s(-1) (at zero ionic strength) effectively in a redox reaction regenerating N2O2(2-) and releasing two NO. This occurs either by electron transfer or, more likely, through radical recombination-dissociation. Each NO so-produced rapidly adds to another N2O2(*-), yielding the N3O3(-) ion, which slowly decomposes at 300 s(-1) to the final N2O + NO2(-) products. The N2O2(*-) radical protonates with pKa = 5.6 +/- 0.3. The neutral HN2O2(*) radical decays by an analogous mechanism but much more rapidly with the apparent second-order rate constant 2k = (1.1 +/- 0.1) x 10(9) M(-1) s(-1). The N2O2(*-) radical shows surprisingly low reactivity toward O2 and O2(*-), with the corresponding rate constants below 1 x 10(6) and 5 x 10(7) M(-1) s(-1). The previously reported rapid dissociation of N2O2(*-) into N2O and O(*-) does not occur. The thermochemistry of HN2O2(*)/N2O2(*-) is discussed in the context of these new kinetic and mechanistic results.     

Reactions of biological oxidants with selenourea: formation of redox active nanoselenium

Reactions of biological oxidizing agents, such as hydroxyl radicals ((*)OH), singlet oxygen ((1)O(2)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) with selenourea were studied. The kinetics of the reactions was followed using time-resolved techniques, and the bimolecular rate constants were determined. In all these reactions, under aerated conditions, elemental red selenium was produced as one of the reaction products. The average size of the selenium particles could be controlled and stabilized in the range of 20-100 nm with the addition of bovine serum albumin (BSA) or sodium dodecyl sulfate (SDS). The particles were characterized by dynamic light scattering studies (DLS), which revealed that the size and distribution of the particles depended mainly on the amount of selenourea undergoing oxidation. Other factors such as the nature of the oxidant and the concentration of the stabilizer also are important in stabilizing the particles. Nanoselenium-reduced ABTS(*-) to colorless ABTS(2-) (ABTS = 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) and oxidized dichlorodihydrofluoresecein diacetate (DCFA) to fluorescent dichlorofluorescein (DCF) indicating its ability to participate in redox and free radical reactions. The reactivity of selenium nanoparticles with these systems varied linearly with the surface area of the particles. The studies demonstrate that selenourea undergoes oxidation with both one-electron and two-electron oxidants to produce elemental selenium, which, on stabilization to nanometer size, exhibits size-dependent redox activity.     

Pulse radiolysis studies on galactose oxidase

Single-Cu-containing galactose oxidase in the GOase(semi) state (Cu(II), no Tyr(*) radical) reacts with pulse radiolysis generated formate radicals CO(2)(*-) to give an intermediate UV-vis spectrum assigned as RSSR(*-), peak at 450 nm (epsilon = 8100 M(-1) cm(-1)). From a detailed kinetic analysis at 450 nm, pH 7.0, the following steps have been identified. First the strongly reducing CO(2)(*-) (-1.9V) reduces GOase(semi) (k(0) > or = 6.5 x 10(8) M(-1) s(-1)) to a species GOase(semi)(*-). This is followed by biphasic reactions (i) GOase(semi)(*-) + GOase(semi) (k(1) = 1.6 x 10(7) M(-1) s(-1)) to give GOase(semi) + P(*-) and (ii) P(*-) + GOase(semi) (k(2) = 6.7 x 10(6) M(-1) s(-1)) to give GOase(semi)RSSR(*-). There are no significant absorbance changes for the formation of GOase(semi)(*-) and P(*-), which are Cu(I) (or related) species. However, GOase(semi)RSSR(*-) has an absorption spectrum which differs significantly from that of GOase(semi). The 450 nm peak is characteristic of an RSSR(*-) radical with two cysteines in close sequence proximity and is here assigned to Cys515-Cys518, which is at the GOase surface and 10.2 A from the Cu. On chemical modification of the RSSR group with HSPO(3)(2-) to give RSSPO(3)H(-) and RS(-), absorbance changes are approximately 50% of those previously observed. The decay of RSSR(*-) (0.17 s(-1)) results in the formation of GOase(red). No RSSR(*-) formation is observed in the reaction of GOase(semi) Tyr495Phe with CO(2)(*-), and a single process giving GOase(red)Tyr495Phe occurs. Similarly in the reaction of GOase(ox) with CO(2)(*-), a single-stage reaction gives GOase(semi).     

Electronic modification of the [Ru(II)(tpy)(bpy)(OH(2))](2+) scaffold: effects on catalytic water oxidation

The mechanistic details of the Ce(IV)-driven oxidation of water mediated by a series of structurally related catalysts formulated as [Ru(tpy)(L)(OH(2))](2+) [L = 2,2'-bipyridine (bpy), 1; 4,4'-dimethoxy-2,2'-bipyridine (bpy-OMe), 2; 4,4'-dicarboxy-2,2'-bipyridine (bpy-CO(2)H), 3; tpy = 2,2';6',2'-terpyridine] is reported. Cyclic voltammetry shows that each of these complexes undergo three successive (proton-coupled) electron-transfer reactions to generate the [Ru(V)(tpy)(L)O](3+) ([Ru(V)=O](3+)) motif; the relative positions of each of these redox couples reflects the nature of the electron-donating or withdrawing character of the substituents on the bpy ligands. The first two (proton-coupled) electron-transfer reaction steps (k(1) and k(2)) were determined by stopped-flow spectroscopic techniques to be faster for 3 than 1 and 2. The addition of one (or more) equivalents of the terminal electron-acceptor, (NH(4))(2)[Ce(NO(3))(6)] (CAN), to the [Ru(IV)(tpy)(L)O](2+) ([Ru(IV)=O](2+)) forms of each of the catalysts, however, leads to divergent reaction pathways. The addition of 1 eq of CAN to the [Ru(IV)=O](2+) form of 2 generates [Ru(V)=O](3+) (k(3) = 3.7 M(-1) s(-1)), which, in turn, undergoes slow O-O bond formation with the substrate (k(O-O) = 3 × 10(-5) s(-1)). The minimal (or negligible) thermodynamic driving force for the reaction between the [Ru(IV)=O](2+) form of 1 or 3 and 1 eq of CAN results in slow reactivity, but the rate-determining step is assigned as the liberation of dioxygen from the [Ru(IV)-OO](2+) level under catalytic conditions for each complex. Complex 2, however, passes through the [Ru(V)-OO](3+) level prior to the rapid loss of dioxygen. Evidence for a competing reaction pathway is provided for 3, where the [Ru(V)=O](3+) and [Ru(III)-OH](2+) redox levels can be generated by disproportionation of the [Ru(IV)=O](2+) form of the catalyst (k(d) = 1.2 M(-1) s(-1)). An auxiliary reaction pathway involving the abstraction of an O-atom from CAN is also implicated during catalysis. The variability of reactivity for 1-3, including the position of the RDS and potential for O-atom transfer from the terminal oxidant, is confirmed to be intimately sensitive to electron density at the metal site through extensive kinetic and isotopic labeling experiments. This study outlines the need to strike a balance between the reactivity of the [Ru═O](z) unit and the accessibility of higher redox levels in pursuit of robust and reactive water oxidation catalysts.     

Singlet oxygen generated from the decomposition of peroxymonocarbonate and its observation with chemiluminescence method

The decomposition of peroxymonocarbonate (HCO(4)(-)) has been investigated by flow-injection chemiluminescence (CL) method. An ultraweak CL was observed during mixing the bicarbonate and hydrogen peroxide solution in organic cosolvent. An appropriate amount of fluorescent organic compounds, such as dichlorofluorescein (DCF), was added to the HCO(4)(-) solution, a strong CL was recorded. Based on studies of the spectrum of fluorescence, CL and UV-vis spectra, electron spin trapping (ESR) technique, mass spectra (MS) and comparison with H(2)O(2)/hypochlorite (ClO(-)) and H(2)O(2)/molybdate (MoO(4)(-)) systems, the CL mechanism was proposed. The reaction is initiated by unimolecular homolysis of the peroxo O-O bond in HO-OCOO(-) molecule. It was suggested that the bond rearrangement within radicals yield superoxide ion (O(2)(*-)). The interaction of superoxide ion with perhydroxyl radical produces singlet oxygen ((1)O(2)). The energy transfers from singlet oxygen to DCF forming an excited energy acceptor (DCF*). Luminescence (lambda(max)=509 nm) was emitted during the relaxation of the energy acceptor to the ground state.     

Selective DNA strand scission with binuclear copper complexes: implications for an active Cu2-O2 species

A homologous series of binuclear copper(II) complexes [Cu(II)(2)(Nn)(Y)(2)](2+) (1-3) (n = 3-5 and Y = (ClO(4))(-) or (NO(3))(-)) were studied to investigate the intermediate(s) responsible for selective DNA strand scission in the presence of MPA/O(2) (MPA = 3-mercaptopropanoic acid). While the N3 complex does not react, the N4 and N5 analogues show comparable activity with strand scission occurring at a single-strand/double-strand junction. Identical reactivity is also observed in the alternate presence of H(2)O(2). Spectroscopic and reactivity studies with [Cu(II)(2)(N4)(Y)(2)](2+) (2) and H(2)O(2) are consistent with DNA oxidation mediated by formation of a side-on peroxodicopper(II) (Cu(2)-O(2)) complex.     

Formation, reactivity, and photorelease of metal bound nitrosyl in [Ru(trpy)(L)(NO)](n+) (trpy = 2,2':6',2'-terpyridine, L = 2-phenylimidazo[4,5-f]1,10-phenanthroline)

Nitrosyl complexes with {Ru-NO} (6) and {Ru-NO} (7) configurations have been isolated in the framework of [Ru(trpy)(L)(NO)] ( n+ ) [trpy = 2,2':6',2'-terpyridine, L = 2-phenylimidazo[4,5- f]1,10-phenanthroline] as the perchlorate salts [ 4](ClO 4) 3 and [ 4](ClO 4) 2, respectively. Single crystals of protonated material [ 4-H (+)](ClO 4) 4.2H 2O reveal a Ru-N-O bond angle of 176.1(7) degrees and triply bonded N-O with a 1.127(9) A bond length. Structures were also determined for precursor compounds of [ 4] (3+) in the form of [Ru(trpy)(L)(Cl)](ClO 4).4.5H 2O and [Ru(trpy)(L-H)(CH 3CN)](ClO 4) 3.H 2O. In agreement with largely NO centered reduction, a sizable shift in nu(NO) frequency was observed on moving from [ 4] (3+) (1953 cm (-1)) to [ 4] (2+) (1654 cm (-1)). The Ru (II)-NO* in isolated or electrogenerated [ 4] (2+) exhibits an EPR spectrum with g 1 = 2.020, g 2 = 1.995, and g 3 = 1.884 in CH 3CN at 110 K, reflecting partial metal contribution to the singly occupied molecular orbital (SOMO); (14)N (NO) hyperfine splitting ( A 2 = 30 G) was also observed. The plot of nu(NO) versus E degrees ({RuNO} (6) --> {RuNO} (7)) for 12 analogous complexes [Ru(trpy)(L')(NO)] ( n+ ) exhibits a linear trend. The electrophilic Ru-NO (+) species [ 4] (3+) is transformed to the corresponding Ru-NO 2 (-) system in the presence of OH (-) with k = 2.02 x 10 (-4) s (-1) at 303 K. In the presence of a steady flow of dioxygen gas, the Ru (II)-NO* state in [ 4] (2+) oxidizes to [ 4] (3+) through an associatively activated pathway (Delta S++ = -190.4 J K (-1) M (-1)) with a rate constant ( k [s (-1)]) of 5.33 x 10 (-3). On irradiation with light (Xe lamp), the acetonitrile solution of paramagnetic [Ru(trpy)(L)(NO)] (2+) ([ 4] (2+)) undergoes facile photorelease of NO ( k NO = 2.0 x 10 (-1) min (-1) and t 1/2 approximately 3.5 min) with the concomitant formation of the solvate [Ru (II)(trpy)(L)(CH 3CN)] (2+) [ 2'] (2+). The photoreleased NO can be trapped as an Mb-NO adduct.     

Antioxidative properties of probucol estimated by the reactivity with superoxide and by electrochemical oxidation

The reaction of probucol with superoxide (O2(*-)) was investigated in acetonitrile using both electron spin resonance (ESR) and electrochemical techniques. The formation of phenoxyl radical was observed during the reaction of probucol with O2(*-) by ESR spectroscopy. The reaction of probucol with O2(*-) in acetonitrile was followed by cyclic voltammetry. With the addition of probucol, the oxidation peak current of O2(*-) decreased concentration dependently. This suggests that probucol reacts with O2(*-), that is, probucol scavenges O2(*-) in acetonitrile. 2,6-Di-tert-butyl-p-benzoquinone was identified as the major product of the reaction of probucol with O2(*-) in acetonitrile. Electrochemical oxidation of probucol was also performed. Probucol gives an irreversible oxidation peak at ca. +1.4 V vs. the saturated calomel electrode in the cyclic voltammogram. Controlled-potential electrolysis was carried out at +1.2 V in a divided cell. 2,6-Di-tert-butyl-p-benzoquinone, 4,4'-dithiobis(2,6-di-tert-butylphenol), and 4,4'-trithiobis(2,6-di-tert-butylphenol) were identified as the products of anodic oxidation. These redox properties of probucol may correlate with the physiological activities.     

The fate of C5' radicals of purine nucleosides under oxidative conditions

The factors that influence the reactivity of C5' radicals in purine moieties under aerobic conditions are unknown not only in DNA, but also in simple nucleosides. 5',8-Cyclopurine lesions are the result of a rapid C5' radical attack to the purine moieties before the reaction with oxygen. These well-known lesions among the DNA modifications were suppressed by the presence of molecular oxygen in solution. Here we elucidate the chemistry of three purine-substituted C5' radicals (i.e., 2'-deoxyadenosin-5'-yl, 2'-deoxyinosin-5'-yl, and 2'-deoxyguanosin-5'-yl) under oxidative conditions using gamma-radiolysis coupled with product studies. 2'-Deoxyadenosin-5'-yl and 2'-deoxyinosin-5'-yl radicals were selectively generated by the reaction of hydrated electrons (e(aq)(-)) with 8-bromo-2'-deoxyadenosine and 8-bromo-2'-deoxyinosine followed by a rapid radical translocation from the C8 to the C5' position. Trapping these two C5' radicals with Fe(CN)6(3-) gave corresponding hydrated 5'-aldehydes in good yields that were isolated and fully characterized. When an oxygen concentration in the range of 13-266 microM (typical oxygenated tissues) is used, the hydrated 5'-aldehyde is accompanied by the 5',8-cyclopurine nucleoside. The formation of 5',8-cyclopurines is relevant in all experiments, and the yields increased with decreasing O2 concentration. The reaction of HO(*) radicals with 2'-deoxyadenosine and 2'-deoxyguanosine under normoxic conditions was also investigated. The minor path of C5' radicals formation was found to be ca. 10% by quantifying the hydrated 5'-aldehyde in both experiments. Rate constants for the reactions of the 2'-deoxyadenosin-5'-yl with cysteine and glutathione in water were determined by pulse radiolysis to be (2.1 +/- 0.5) x 10(7) and (4.9 +/- 0.6) x 10(7) M(-1) s(-1) at 22 degrees C, respectively.     

Stepwise synthesis of [Ru(trpy)(L)(X)](n+) (trpy = 2,2':6',2' '-terpyridine; L = 2,2'-dipyridylamine; X = Cl-, H2O, NO2-, NO+, O2-). Crystal structure, spectral, electron-transfer, and photophysical aspects

Ruthenium-terpyridine complexes incorporating a 2,2'-dipyridylamine ancillary ligand [Ru(II)(trpy)(L)(X)](ClO(4))(n) [trpy = 2,2':6',2' '-terpyridine; L = 2,2'-dipyridylamine; and X = Cl(-), n = 1 (1); X = H(2)O, n = 2 (2); X = NO(2)(-), n = 1 (3); X = NO(+), n = 3 (4)] were synthesized in a stepwise manner starting from Ru(III)(trpy)(Cl)(3). The single-crystal X-ray structures of all of the four members (1-4) were determined. The Ru(III)/Ru(II) couple of 1 and 3 appeared at 0.64 and 0.88 V versus the saturated calomel electrode in acetonitrile. The aqua complex 2 exhibited a metal-based couple at 0.48 V in water, and the potential increased linearly with the decrease in pH. The electron-proton content of the redox process over the pH range of 6.8-1.0 was calculated to be a 2e(-)/1H(+) process. However, the chemical oxidation of 2 by an aq Ce(IV) solution in 1 N H(2)SO(4) led to the direct formation of corresponding oxo species [Ru(IV)(trpy)(L)(O)](2+) via the concerted 2e(-)/2H(+) oxidation process. The two successive reductions of the coordinated nitrosyl function of 4 appeared at +0.34 and -0.34 V corresponding to Ru(II)-NO(+) --> Ru(II)-NO* and Ru(II)-NO* --> Ru(II)-NO(-), respectively. The one-electron-reduced Ru(II)-NO* species exhibited a free-radical electron paramagnetic resonance signal at g = 1.990 with nitrogen hyperfine structures at 77 K. The NO stretching frequency of 4 (1945 cm(-1)) was shifted to 1830 cm(-1) in the case of [Ru(II)(trpy)(L)(NO*)](2+). In aqueous solution, the nitrosyl complex 4 slowly transformed to the nitro derivative 3 with the pseudo-first-order rate constant of k(298)/s(-1) = 1.7 x 10(-4). The chloro complex 1 exhibited a dual luminescence at 650 and 715 nm with excited-state lifetimes of 6 and 1 micros, respectively.     

Electronic properties, redox behavior, and interactions with H2O2 of pH-sensitive hydroxyphenyl-1,2,4-triazole-based oxovanadium(V) complexes

The syntheses and spectroscopic characterization of two 1,2,4-triazole-based oxovanadium(V) complexes are reported: 1- [VO2L1]- and 2 [(VOL2)2(OMe)2] (where H2L1 = 3-(2'-hydroxyphenyl)-5-(pyridin-2' '-yl)-1H-1,2,4-triazole, H3L2 = bis-3,5-(2'-hydroxyphenyl)-1H-1,2,4-triazole). The ligand environment (N,N,O vs O,N,O) is found to have a profound influence on the properties and reactivity of the complexes formed. The presence of the triazolato ligand allows for pH tuning of the spectroscopic and electrochemical properties, as well as the interaction and stability of the complexes in the presence of hydrogen peroxide. The vanadium(IV) oxidation states were generated electrochemically and characterized by UV-vis and EPR spectroscopies. For 2, under acidic conditions, rapid exchange of the methoxide ligands with solvent [in particular, in the vanadium(IV) redox state] was observed.     

Direct observation of the one-electron reduction of methyl viologen mediated by the CO2 radical anion during TiO2 photocatalytic reactions

The one-electron reduction of methyl viologen (MV(2+)) mediated by the carbon dioxide radical anion (CO(2)(*-)) during photocatalytic reactions in a colloidal TiO(2) aqueous solution (pH 2) has been investigated by time-resolved absorption spectroscopy. The formation of MV(*+) generated from the one-electron reduction reaction with CO(2)(*-), which is generated from the one-electron oxidation reactions with the photogenerated holes (h(+)), was directly observed. The spectral features of the photogenerated charge carriers and the kinetic analysis of the formation process of MV(*+) revealed that the CO(2)(*-), desorbed from the surface, reacts with MV(2+) via a homogeneous electron-transfer process in the bulk solution.     

Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate

Spin trapping using a nitrone and electron paramagnetic resonance (EPR) spectroscopy is commonly employed in the identification of transient radicals in chemical and biological systems. There has also been a growing interest in the pharmacological activity of nitrones, and there is, therefore, a pressing need to develop nitrones with improved spin trapping properties and controlled delivery in cellular systems. The beta-cyclodextrin (beta-CD)-cyclic nitrone conjugate, 5-N-beta-cyclodextrin-carboxamide-5-methyl-1-pyrroline N-oxide (CDNMPO) was synthesized and characterized. 1-D and 2-D NMR show two stereoisomeric forms (i.e., 5S- and 5R-) for CDNMPO. Spin trapping using CDNMPO shows distinctive EPR spectra for superoxide radical anion (O2(*-)) compared to other biologically relevant free radicals. Kinetic analysis of O2(*-) adduct formation and decay using singular value decomposition and pseudoinverse deconvolution methods gave an average bimolecular rate constant of k = 58 +/- 1 M(-1) s(-1) and a maximum half-life of t(1/2) = 27.5 min at pH 7.0. Molecular modeling was used to rationalize the long-range coupling between the nitrone and the beta-CD, as well as the stability of the O2(*-) adducts. This study demonstrates how a computational approach can aid in the design of spin traps with a relatively high rate of reactivity to O2(*-), and how beta-CD can improve adduct stability via intramolecular interaction with the O2(*-) adduct.     

Validation of a new method using the reactivity of electrogenerated superoxide radical in the antioxidant capacity determination of flavonoids

This article lays out a new method to measure the antioxidant capacity of some flavonoids. The methodology developed is based on the kinetics of the reaction of the antioxidant substrate with the superoxide radical (O(2)(*-)). A cyclic voltammetric technique was used to generate O(2)(*-) by reduction of molecular oxygen in aprotic media. In the same experiment the consumption of the radical was directly measured by the anodic current decay of the superoxide radical oxidation in the presence of increasing concentrations of antioxidant substrate. The method was statistically validated on flavonoid monomers and on the standard antioxidants: trolox, ascorbic acid and phloroglucinol. The linear correlations between the anodic current of O(2)(*-) and the substrate concentration allowed the determination of antioxidant index values expressed by the substrate concentration needed to consume 30% (AI(30)) and 50% (AI(50)) of O(2)(*-) in given conditions of oxygen concentration and scanning rate. The fidelity of the method was examined intraday and interlaboratories.     

Cu2+ and Cu+ bathocuproine disulfonate complexes promote the oxidation of the ROS-detecting compound dichlorofluorescin (DCFH)

The water-soluble Cu⁺chelator bathocuproine disulfonate (BCS) is widely used to quantify Cu⁺ or detect Cu⁺ formation in Cu²⁺-initiated oxidation reactions. The dichlorofluorescin (DCFH) assay is commonly used to monitor free radical reactions, reactive oxygen species (ROS), or reactive nitrogen species (RNS). Upon oxidation the non-fluorescent DCFH is converted into the fluorescent compound dichlorofluorescein (DCF). In the present communication we show that the Cu⁺ reagent BCS strongly facilitated the oxidation of DCFH in the presence of Cu²⁺ or Cu⁺. In contrast, 2,2′-dipyridyl (DP), which is also a Cu⁺-complexing reagent, but not as well known and therefore not as commonly used as BCS, did not cause any oxidative modification of DCFH in the presence of Cu²⁺ or Cu⁺. We therefore recommend that DP should be used instead of BCS to complex Cu⁺ in reactions which are initiated by Cu²⁺ and when ROS/RNS are analyzed by the DCFH oxidation assay.     

Structural,spectroscopic, and redox consequences of a central ligand in the FeMoco of nitrogenase: a density functional theoretical study

Broken symmetry density functional and electrostatics calculations have been used to shed light on which of three proposed atoms, C, N, or O, is most likely to be present in the center of the FeMoco, the active site of nitrogenase. At the Mo(4+)4Fe(2+)3Fe(3+) oxidation level, a central N(3-) anion results in (1) calculated Fe-N bond distances that are in very good agreement with the recent high-resolution X-ray data of Einsle et al.; (2) a calculated redox potential of 0.19 eV versus the standard hydrogen electrode (SHE) for FeMoco(oxidized) + e(-) --> FeMoco(resting), in good agreement with the measured value of -0.042 V in Azotobacter vinelandii; and (3) average M?ssbauer isomer shift values (IS(av) = 0.48 mm s(-1)) compatible with experiment (IS(av) = 0.40 mm s(-1)). At the more reduced Mo(4+)6Fe(2+)1Fe(3+) level, the calculated geometry around a central N(3-) anion still correlates well with the X-ray data, but the average M?ssbauer isomer shift value (IS(av) = 0.54 mm s(-1)) and the redox potential of -2.21 eV show a much poorer agreement with experiment. These calculated structural, spectroscopic, and redox data indicate the most likely iron oxidation state for the resting FeMoco of nitrogenase to be 4Fe(2+)3Fe(3+). At this favored oxidation state, oxygen or carbon coordination leads to (1) Fe-O distances in poor agreement and Fe-C distances in good agreement with experiment and (2) calculated redox potentials of +0.97 eV for O(2-) and -1.31 eV for C(4-). The calculated structural parameters and/or redox data suggest either O(2-) or C(4-) is unlikely as a central anion.     

Indirect detection of photosensitizer ex vivo

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0-5 microg x ml(-1)) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg x kg(-1) iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg x kg(-1)), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.     

Photosensitized paraquat-induced structural alterations and free radical mediated fragmentation of serum albumin

Photosensitization of paraquat with photosynthetically active radiations (PAR) induced substantial production of both the hydroxyl radicals (*OH) and superoxide anions (O(2)(*-)) under in vitro conditions. Addition of transition metal ion, Cu (II) enhanced the paraquat-induced *OH radical production by 1.8-fold. Treatment of bovine serum albumin (BSA) with photosensitized paraquat resulted in a dose dependent fragmentation of protein. The quantitative analysis revealed the release of 73 microM acid soluble amino groups and 450 microM carbonyl groups from treated albumin at the highest albumin-paraquat molar ratio of 1:8 in presence of 200 microM Cu (II). The results with the free radical quenchers such as mannitol and superoxide dismutase (SOD) clearly reflected the involvement of *OH radicals in protein fragmentation process. The fluorescence data revealed significantly higher binding of paraquat with serum albumin. The binding constants (K(a)) and binding capacity (n) of albumin for paraquat were determined to be 3.4 x 10(5) l/mole and 12.9, respectively. Fluorescence emission spectra exhibited significant quenching of the intrinsic fluorescence of albumin upon addition of paraquat at increasing molar ratios. This is attributed to induced conformational changes in protein structure upon paraquat interaction at specific sites on albumin molecule. Most likely, the alkyl group transfers occur from N1 and/or N1' positions of paraquat to the electron rich sites at critical amino acid residues on treated protein. At higher paraquat concentrations, the albumin-paraquat interaction resulted in adduct formation with concurrent protein alkylation and free radical mediated fragmentation. Thus, on the basis of these results, the paraquat-protein interaction leading to alkylation, structural alterations and/or fragmentation of biological macromolecules has been suggested as an important factor for agrochemical-induced toxicity.     

Rapid cross-disproportionation between superoxometal ions and acylperoxyl radicals

A superoxochromium complex Cr(aq)OO(2+) reacts with acetylperoxyl radicals, CH(3)C(O)OO(*), with a rate constant of 1.49 x 10(8) M(-1) s(-1). The kinetics were determined by laser flash photolysis, using an organocobalt complex as a radical precursor and ABTS(*-) as a kinetic probe. The initial step is believed to involve radical coupling at the remote oxygen of Cr(aq)OO(2+), followed by elimination of O(2) and formation of CH(3)COOH and Cr(V)(aq)O(3+). The latter disproportionates and ultimately yields Cr(aq)(3+) and HCrO(4)(-).