Use of Raman spectroscopy for the identification of radical-mediated damages in human serum albumin

Damages induced by free radicals on human serum albumin (HSA), the most prominent protein in plasma, were investigated by Raman spectroscopy. HSA underwent oxidative and reductive radical stress. Gamma-irradiation was used to simulate the endogenous formation of reactive radical species such as hydrogen atoms (^•H), solvated electrons (e_aq⁻) and hydroxyl radicals (^•OH). Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and specific damages occurring at sensitive amino acid sites. In particular, the analysis of the S–S stretching region suggested the radical species caused modifications in the 17 disulphide bridges of HSA. The concomitant action of e_aq⁻ and ^•H atoms caused the formation of cyclic disulphide bridges, showing how cystine pairs act as efficient interceptors of reducing species, by direct scavenging and electron transfer reactions within the protein. This conclusion was further confirmed by the modifications visible in the Raman bands due to Phe and Tyr residues. As regards to protein folding, both oxidative and reductive radical stresses were able to cause a loss in α-helix content, although the latter remains the most abundant secondary structure component. β-turns motifs significantly increased as a consequence of the synergic action of e_aq⁻ and ^•H atoms, whereas a larger increase in the β-sheet content was found following the exposure to ^•OH and/or ^•H attack.     

7-Ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone (echinochrome A): a DFT study of the antioxidant mechanism

Quantum chemical calculations and the conformational analysis of dianions, radical dianions, disodium salts, and radicals of disodium salts of 7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone (echinochrome A) were carried out at the (U)B3LYP/6-31G(d) and (U)B3LYP/6-311G(d) levels of theory. The heats of reactions of the hydroperoxyl radical (HOO^·) with the isomers of dianions and disodium salts of echinochrome A with the lowest Gibbs free energies were estimated. All reactions of these isomers of dianions and disodium salts of echinochrome A with HOO^· in the gas phase are exothermic. The isomer of the dianion of echinochrome A with the lowest Gibbs free energy, which is formed by the heterolysis of the 2β- and 6β-OH groups, is the more effective antioxidant than the isomer of the 2,6-disodium salt with the lowest Gibbs free energy.     

Synthesis and characterization of stable thiazolylazo anion radical complexes of ruthenium(II)

Two stable thiazolylazo anion radical complexes of ruthenium(II), [Ru(L1^•−)(Cl)(CO)(PPh₃)₂] (1) and [Ru(L2^•−)(Cl)(CO)(PPh₃)₂] (2) (where L1 = 2′-Thiazolylazo-2-imidazole and L2 = 4-(2′-Thiazolylazo)-1-n-hexadecyloxy-naphthalene), have been synthesized and characterized by spectroscopic and electrochemical techniques. The radical nature of the complexes has been confirmed from their room temperature magnetic moments and X-band ESR spectra. The radical complexes display a moderately intense (ε ~ 10⁴ M⁻¹ cm⁻¹) and relatively broad band in 430–460 nm region. In the microcrystalline state, complexes (1) and (2) display strong ESR signals at g = 1.951 and g = 1.988, respectively. In CH₂Cl₂ solution, complexes (1) and (2) show a quasireversible one-electron response near −0.64 and −0.59 V, respectively, versus Ag/AgCl due to the radical redox couple [Ru^II(L)(Cl)(CO)(PPh₃)₂]/[Ru^II (L^•−)(Cl)(CO)(PPh₃)₂].     

Synthesis and properties of paramagnetic derivatives of linear and fused polyaromatic compounds

Paramagnetic derivatives of tolan, anthracene, and terphenyl of the A−Sp−R^• type, where A is an aromatic group, R^• is a stable radical center, and Sp is a spacer, were synthesized. The electronic absorption and luminescence spectra of these compounds were examined. The introduction of the paramagnetic dihydroimidazole fragment causes a decrease (10–500-fold) in the quantum yield of luminescence compared to emission of the individual aromatic luminophore. The presence of a radical center (R^•) in the (alkane)^•+/−•A−Sp−R^• radical-ionic pair leads to the disappearance or fast (nanoseconds) damping of the magnetic effect of luminescence arising upon recombination of these pairs.     

Transient species and its properties of melatonin

Reactive oxygen species (ROS) are generated dur- ing radiation, respiratory burst, normal metabolic processes and so on. There are enzymatic and non-enzymatic antioxidants such as superoxide dis- mutase (SOD), glutathione peroxidase (GSH-Px), vi- tamin E (VE) and carotenoids that can either inhibit or repair the ROS-induced damage. ROS is essential to maintain physiological homeostasis. However, exces- sive ROS give rise to oxidative damage to proteins, lipids and DNA which related t…     

Novel C<Subscript>β</Subscript>–C<Subscript>γ</Subscript> Bond Cleavages of Tryptophan-Containing Peptide Radical Cations

In this study, we observed unprecedented cleavages of the C_β–C_γ bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M^•+) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M – 116]⁺ ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent C_β–C_γ bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH – 43]⁺ and [WGGGH – 116]⁺, obtained from the CID of [LGGGH]^•+ and [WGGGH]^•+, respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind]^•-2), in agreement with the CID data for [WGGGH]^•+ and [W_1-CH3GGGH]^•+; replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from C_β–C_γ bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the C_β–C_γ bond and, therefore, decreases the dissociation energy barrier dramatically.     

Synthesis and anion recognition of neutral receptors based on multiamide calix[4]arene

Two multiamide calix[4]arenes (5, 6) were synthesized and characterized by NMR, MS and elemental analysis. The binding properties of receptors with some anions (π-O₂NPhOPO₃²⁻, π-O₂NPhO⁻, H₂PO₄⁻, Ac⁻, Cl⁻, Br⁻ and I⁻) were studied by UV-Vis spectra. The results indicate that the tetraamide calix[4]arenes (5, 6) have a good selectivity to the anions containing aromatic ring (π-O₂NPhOPO₃ ²⁻, π-O₂NPhO⁻). The 1 : 1 complexes between host and guest were formed through multiple hydrogen bonding and π-π interactions. The hosts 5 and 6 also show a definite binding ability for the anions (H₂PO₄⁻, Ac⁻, Cl⁻) that have no ultraviolet absorption, which provides a simple method of spectrum detection for these anions.     

Anti- and pro-oxidant properties of sodium 2-mercaptoethane sulphonate (Mesna)

The ^•OH and the NO₂ ^• radicals generated pulse radiolytically in N₂O-saturated aqueous solution at pH 8–8.5 oxidize Mesna to form the corresponding thiyl radicals which on reaction with thiolate ions form an RSSR^{• −} type of transient with λ_max = 420 nm. The rate constants for the formation of these transients were determined. In the absence of O₂ at pH=6, the RS^• radicals formed show an absorption maximum at 360 nm and an ε=200±50 dm³ mol⁻¹ cm⁻¹. The rate constant k (^•OH+RSH) was 6×10⁹ dm³ mol⁻¹ s⁻¹ as determined from competition kinetics. In the presence of O₂ the Mesna thiyl radical was seen to rapidly add oxygen to form an RSOO^• type of species with λ_max = 535 nm, ε=700±50 dm³ mol⁻¹ cm⁻¹ and k (RS^•+O₂)=1.3×10⁸ dm³ mol⁻¹ s⁻¹. Both the RS^• and the RSOO^• radicals formed by the oxidation of Mesna were able to abstract H-atoms from ascorbate ions and k(RS^• +AH⁻)=~k(RSOO^•+AH⁻)=~6−7×10⁸ dm³ mol⁻¹ s^−1-. Moderately strong oxidants like CCl₃OO^• and the (CH₃)₃CO^• radicals, having a reduction potential of +1.4−1.6 V vs NHE were unable to oxidize Mesna. The results thus reflect on the pro- and anti-oxidant properties of Mesna.     

Kinetics of the ?OH-radical initiated reactions of acetic acid and its deuterated isomers

Kinetics of the ^•OH-initiated reactions of acetic acid and its deuterated isomers have been investigated performing simulation chamber experiments at T = 300 ± 2 K. The following rate constant values have been obtained (± 1σ, in cm³ molecule⁻¹ s⁻¹): k ₁(CH₃C(O)OH + ^•OH) = (6.3 ± 0.9) × 10⁻¹³, k ₂(CH₃C(O)OD + ^•OH) = (1.5 ± 0.3) × 10⁻¹³, k ₃(CD₃C(O)OH + ^•OH) = (6.3 ± 0.9) × 10⁻¹³, and k ₄(CD₃C(O)OD + ^•OH) = (0.90 ± 0.1) × 10⁻¹³. This study presents the first data on k ₂(CH₃C(O)OD + ^•OH). Glyoxylic acid has been detected among the products confirming the fate of the ^•CH₂C(O)OH radical as suggested by recent theoretical studies.     

Determination of thiocyanate ions at nanolevel in real samples using coated graphite electrode based on synthesised macrocyclic Zn(II) complex

The electrode characteristics and selectivities of PVC-based thiocyanate selective polymeric membrane electrode (PME) incorporating the newly synthesized zinc complex of 6,7:14,15-Bzo₂-10,11-(4-methylbenzene)-[15]-6,8,12,14-tetraene-9,12-N₂-1,5-O₂ (I ₁ ) and zinc complex of 6,7:14,15-Bzo₂-10,11-(4-methylbenzene)-[15]-6,14-diene-9,12-dimethylacrylate-9,12-N₂-1,5-O₂ (I ₂ ) are reported here. The best response was observed with the membrane having a composition of I₂:PVC:o-NPOE:HTAB in the ratio of 6:33:59:2 (w/w; milligram). This electrode exhibited Nernstian slope for thiocyanate ions over working concentration range of 4.4 × 10⁻⁷ to 1.0 × 10⁻² mol L⁻¹ with detection limit of 2.2 × 10⁻⁷ mol L⁻¹. The performance of this electrode was compared with coated graphite electrode (CGE), which showed better response characteristics w.r.t Nernstian slope 59.0 ± 0.2 mV decade⁻¹ activity, wide concentration range of 8.9 × 10⁻⁸ to 1.0 × 10⁻² mol L⁻¹ and detection limit of 6.7 × 10⁻⁸ mol L⁻¹. The response time for CGE and PME was found to be 8 and 10 s, respectively. The proposed electrode (CGE) was successfully applied to direct determination of thiocyanate in biological and environmental samples and also as indicator electrode in potentiometric titration of SCN⁻ ion.     

Electrochemistry of bis(indenyl)dimethylzirconium complex—the precursor of the olefin polymerization catalyst

The reduction in THF and oxidation in CH₂Cl₂ of the bent-sandwich complex (η⁵-lnd)₂ZrMe₂ (1) (Ind=C₉H₇, indenyl) were studied by cyclic voltammetry. Complex1 in THF undergoes one-electron reduction to radical anion1 ⁻, which partially decomposes with the liberation of the Ind⁻ anion. Even at −45°C the one-electron oxidation leads to the formation of an unstable 15-electron radical cation undergoing fast heterolytic decomposition to the Me^• radical and (η⁵-lnd)₂ZrMe² cation, which is the key reaction center in the catalytic polymerization of olefins. Comparative analysis of electron-transfer-induced transformations of bent-sandwich dimethyl and dichloride zirconocenes of the general formula L₂ZrX₂ (L=η⁵-lnd, η⁵-Cp: X=Xl, Me) was performed. The material of the paper was first reported at the 195th Meeting of the Electrochemical Society (see Ref. 1). Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 59–62, January, 2000.     

Electron paramagnetic resonance (EPR) study of γ-radiation-induced radicals in 1,3,5-trithiane and its derivatives

Radicals formed in γ-irradiated 1,3,5-trithiane (TT) and its three derivatives, α- and β-2,4,6-trimethyl-1,3,5-trithiane (α-TMT and β-TMT), and 2,4,6-trimethyl-2,4,6-triphenyl-1,3,5-trithiane (TMTPT), were studied by the electron paramagnetic resonance (EPR) method in the solid state. The sulfur radical cations (>S^+•) were identified in all compounds at 77 K. In TT and its two derivatives, α-TMT and β-TMT, the >S^+• decay via deprotonation-forming C-centered radicals. Further increase of temperature up to 293 K results in the appearance of thiyl-type radicals (RS^•). In TMTPT, the >S^+• are stable up to 250 K. They formed the intermolecularly three-electron-bonded dimeric radical cations (S∴S)⁺ while RS^• radicals were not observed. Some of the radical assignments and their EPR parameters (g and a hyperfine splittings) obtained support from the DFT calculations.     

Stereochemical effects during [M-H]− dissociations of epimeric 11-OH-17β-estradiols and distant electronic effects of substituents at C(11) position on gas phase acidity

The affinity of estradiol derivatives for the estrogen receptor (ER) depends strongly on nature and stereochemistry of substituents in C₍₁₁₎ position of the 17β-estradiol (I). In this work, the stereochemistry effects of the 11α-OH-17β-estradiol (III_α) and 11β-OH-17β-estradiol (III_β) were investigated using CID experiments and gas-phase acidity (ΔH_acid^∘) determination. The CID experiments showed that the steroids decompose via different pathways involving competitive dissociations with rate constants depending upon the α/β C₍₁₁₎ stereochemistry. It was shown that the fragmentations of both deprotonated [III_α-H]⁻ and [III_β-H]⁻ epimers were initiated by the deprotonation of the most acidic site, i.e. the phenolic hydroxyl at C₍₃₎. This view was confirmed by H/D exchange and double resonance experiments. Furthermore, the ΔH_acid^∘ of both epimers (III_α and III_β), 17β-estradiol (I), and 17-desoxyestradiol (II) was determined using the extended Cooks’ kinetic method. The resulting values allowed us to classify steroids as a function of their gas-phase acidity as follows: (III_β)≫(II)>(I)>(III_α). Interestingly, the α/β C₍₁₁₎ stereochemistry appeared to influence strongly the gas-phase acidity. This phenomenon could be explained through stereospecific proton interaction with π-orbital cloud of A ring, which was confirmed by theoretical calculation.     

Formation of p-terphenyl excited states in the ionic liquid methyltributylammonium bis[(trifluoromethyl)sulfonyl]imide: pulse radiolysis study

Solutions of 80 mM benzophenone (BP) and up to 14 mM p-terphenyl (TP) in the ionic liquid methyltributylammonium bis[(trifluoromethyl)sulfonyl]imide (R₄NNTf₂) have been investigated by nanosecond pulse radiolysis. The resulting transient absorption spectra of pulse-irradiated argon saturated solutions correspond to the formation of several intermediates derived from BP and TP: benzophenone radical anion [(C₆H₅)₂CO]^•− (BP^•−) converted after ~20 μs into ketyl radicals (C₆H₅)₂C^•OH (BP^•H), a hydrogen adduct to the phenyl ring of benzophenone C₆H₅COC₆H₆^•, p-terphenyl triplet excited state ³TP*, and traces of TP radical ions. ³TP* was formed in two steps, the first immediately during the pulse and the second in pseudo-first order process with a second order reaction rate constant calculated from TP concentration dependence: k = ~2 × 10⁸ dm³ mol⁻¹ s⁻¹.     

The modes of complexation of benzimidazole with aqueous β-cyclodextrin explored by phase solubility, potentiometric titration, 1H-NMR and molecular modeling studies

The results of rigorous modeling of phase solubility diagrams, pH solubility profiles and potentiometric titrations revealed the following for benzimidazole (BZ) and BZ/β-CD complexation in aqueous solution: (a) the pK _a value of BZ estimated at 5.66 ± 0.08 was reduced to 5.33 ± 0.06 in the presence of 15 mM β-CD at 25 °C, thus indicating inclusion complex formation; (b) BZ forms soluble 1:1 and 2:1 BZ/β-CD complexes with complex formation constants K ₁₁ = 104 ± 8 M⁻¹ and K ₂₁ = 16 ± 6 M⁻¹; (c) protonated BZ forms only 1:1 complex with K ₁₁ = 42 ± 12 M⁻¹; (d) ¹H-NMR studies in D₂O showed significant upfield chemical shift displacements for inner cavity β-CD protons indicating inclusion complex formation, while (e) Molecular modeling of BZ-β-CD interactions in water clearly indicated complete inclusion of one BZ molecule into the β-CD cavity.     

Cis-coordination of the chromate anions in helical Co/Ni block-type polymeric systems. Structural and spectroscopic characteristics of catena(μ-CrO4-O,O′)[Co(HIm)3H2O] and catena(μ-CrO4-O,O′)[Co0.43Ni0.57(HIm)3H2O]

Two polymeric complexes: catena(μ-CrO₄-O,O′)[Co(HIm)₃H₂O] (1) and catena(μ-CrO₄-O,O′)[Co_0.43Ni_0.57(HIm)₃H₂O] (2) (where HIm=imidazole) with a cis-bridging coordination mode of the CrO₄ ²⁻ anion have been synthesized and characterized by X-ray and spectroscopic methods. These crystals were isolated from nine systems of varying reagent molar ratios and three excluding anions: Cl⁻, NO₃ ⁻ and SO₄ ²⁻ exclusively as mer [M(HIm)₃O₃]-type isomers. The unit cell of these isostructural complexes (monoclinic crystal system P2₁ /n) contains two independent helixes, left- and right handed, stabilized by intrahelical and interhelical hydrogen bonding and π–π interaction between pairs of the imidazole rings from neighbouring helixes. The Raman spectra at 77 K of 1 and 2 deconvoluted into lorentzian components revealed the block-type polymeric structure of the complexes. Moreover, the solution studies at millimolar concentrations of 1 and 2 indicated their complete decomposition in water. Four K electronic spectral analysis of the crystals (band deconvolution into gaussian components) enhanced with the data obtained in the polarized light allowed for assignment of the bands to the respective d–d transition (D_4h symmetry). It was found that the metallic centres are independently absorbing species, which supports the suggestion of a block-type structure of the polymers. The respective crystal field parameters for Co and Ni were calculated.     

A CIDEP study of photolysis of duroquinone/hydrogen-donor homogeneous and triton micelle solutions

The CIDEP spectra of transient radicals during photolysis of the duroquinone (DQ)/ethylene glycol (EG) system in acid, basic, and micellar environments were measured with a home-made highly time-resolved ESR spectrometer. In the DQ/EG homogeneous solution, the enhanced emissive CIDEP signal of the neutral durosemiquinone radical DQH^• was observed. When the DQ/EG solution at pH 9 or the DQ/EG/TX-100/H₂O micelle system was photolyzed, the CIDEP signal of the duroquinone anion radical (DQ^•−) was obtained. When the DQ/EG solution at pH 2.5 was irradiated, the CIDEP signal of DQH^• appeared. These experimental results indicate that the neutral radical DQH^• was formed by proton transfer from EG to ³DQ*, and that DQ^•− was formed by dissociation of DQH^• accompanying polarization transfer.     

Structure of complex salts [Co(NH3)6][Rh(NO2)6] and [Co(NH3)6][(NO2)3Rh(μ-NO2)1+x(μ-OH)2?xRh(NO2)3]·(2-x)(H2O), x = 0.17

The structures of two salts [Co(NH₃)₆][Rh(NO₂)₆] (I) and [Co(NH₃)₆][(NO₂)₃Rh(μ-NO₂)_1+x (μ-OH)_2−x Rh(NO₂)₃]·(2−x)(H₂O), x = 0.17 (II) are solved. Single crystals of the salts are obtained by the counter diffusion method through the gel of aqueous solutions of [Co(NH₃)₆]Cl₃ and Na₃[Rh(NO₂)₆]. The structure of [Co(NH₃)₆][Rh(NO₂)₆] is consistent with the diffraction data for a polycrystalline sample of poorly soluble fine salt formed in the exchange reaction between aqueous solutions of [Co(NH₃)₆]Cl₃ and Na₃[Rh(NO₂)₆]. The structure of [Co(NH₃)₆][(NO₂)₃Rh(μ-NO₂)_1+x (μ-OH)_2−x Rh(NO₂)₃]·(2−x)(H₂O), x = 0.17 exhibits the stabilizing effect of a large cation in the formation of novel, unknown previously coordination ions: [(NO₂)₃Rh(μ-NO₂)(μ-OH)₂Rh(NO₂)₃]³⁻ and [(NO₂)₃Rh(μ-NO₂)₂(μ-OH)Rh(NO₂)₃]³⁻.     

Voltammetric response and electrochemical properties of the O2/O 2 •− couple in acetone

Voltammetric responses of the O₂/O ₂ ^•− couple as a function of aprotic solvents are investigated. The results show that acetone is a suitable solvent for examining the electrode reaction of the O₂/O ₂ ^•− couple. Studying the electrochemical behavior of the O₂/O ₂ ^•− couple at different electrodes in acetone by cyclic voltammetry suggests that the reversibility of the O₂/O ₂ ^•− couple and the redox peak currents at glassy carbon are better than those at gold and platinum. The kinetic parameters (electron transfer rate constant k ₀ and cathodic transfer coefficient α) of the oxygen reduction to superoxide ion (O ₂ ^•− ) are evaluated using normal-pulse voltammetry. The obtained values of k ₀ (cm/s) and α are (1.95 ± 0.05) × 10⁻⁴ and 0.34 ± 0.01, respectively. The scavenging activities of ascorbic acid and bilirubin are tested and the experimental results confirm that ascorbic acid is a better scavenger toward O ₂ ^•− . Published in Russian in Elektrokhimiya, 2007, Vol. 43, No. 2, pp. 188–194. The text was submitted by the authors in English.