The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation.

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.

Fe(III) - Sulfide interaction in globins: Characterization and quest for a putative Fe(IV)-sulfide species.

The present study reports findings regarding the contrast between H2S interaction with bovine hemoglobin (Hb) and horse heart myoglobin (Mb), in terms of binding and dissociation kinetics, affinities, and mechanism. At pH9.5, oxidation of ferric-sulfide adducts in presence of no free sulfide, using hexachloroiridate as oxidant is examined using stopped-flow UV-vis, EPR, vibrational spectroscopy and mass spectrometry. Oxidation of the ferric-sulfide adduct in such conditions occurs with a putative unstable Fe(IV)-sulfide adduct as intermediate that finally leads to a paramagnetic ferric species with distinct EPR features. As detected by MS spectrometry, this final species appears to be a truncated form of globin at a distinct Tyr. In case of Hb, only ß-chain is truncated at Tyr144.

Comparative studies of reaction of cobalamin (II) and cobinamide (II) with sulfur dioxide.

The kinetics of reactions of cobalamin (II) and cobinamide (II) with sulfur dioxide was studied by UV-visible (UV-vis) spectroscopy. Reaction results in oxidation of Co(II) center and involves two aquated SO2 moieties. The final product is suggested to be complex Co(III)-S2O 4•- . The absence of corrin ring modifications during the reactions was proved.

Chlorite reactivity with myoglobin: Analogy with peroxide and nitrite chemistry?

Stopped-flow UV-vis data allow for the first time direct spectroscopic detection of a ferryl species during the reaction of met myoglobin (Mb) with chlorite, analogous to what is observed in the reaction with peroxides. Ferryl is also observed in the reaction of oxy Mb+chlorite. A pathway involving Fe-O-O-ClO2 is explored by analogy with the Fe-O-O-NO and Fe-O-O-NO2 previously proposed as intermediates in the reactions of oxy globins with nitric oxide and nitrite, respectively. However, Fe-O-O-ClO2 is not detectable in these stopped-flow experiments and is in fact, unlike its nitrogenous congeners, predicted by density functional theory (DFT) to be impossible for a heme complex. Deoxy Mb reacts with chlorite faster than met - suggesting that, unlike with hydrogen peroxide (with which deoxy Mb reacts slower than met), binding of chlorite to the heme is not a rate-determining step (hence, most likely, an outer-sphere electron transfer mechanism); to correlate this, a Fe-O-Cl-O adduct was not observed experimentally for the met or for the deoxy reactions - even though prior DFT calculations suggest it to be feasible and detectable.

Achillea schurii Flowers: Chemical, Antioxidant, and Antimicrobial Investigations.

This study aims to evaluate the phenolic profile, and antioxidant and antimicrobial activity of Achillea schurii Sch.-Bip., an endemic species from Romania that has not been investigated yet. The chromatographic profile of the phenolic components was obtained using the HPLC-MS method, while the total polyphenol, flavonoid, caffeic acid derivative contents were quantified using spectrophotometric methods. The antioxidant activity was evaluated using different methods: DPPH radical scavenging, hemoglobin ascorbate peroxidase activity inhibition (HAPX), inhibition of lipid peroxidation catalyzed by cytochrome c, and direct detection of plant-derived free radicals using electron paramagnetic resonance (EPR). The antimicrobial test was performed using the disk diffusion assay. The phenolic profile has revealed high amounts of isoquercitrin, rutin, luteolin, and apigenin. The A. schurii extract exhibited a good antioxidant capacity, and high phenolic contents (76.93 mg/g polyphenols, 18.61 mg/g flavonoids and 41.48 mg/g caffeic acid derivatives, respectively). The antimicrobial tests reveal a remarkable inhibitory activity against Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhimurium. Considering the above, A. schurii may be deemed to offer good perspectives for pharmaceutical and industrial applications.

Antioxidant activity evaluation by physiologically relevant assays based on haemoglobin peroxidase activity and cytochrome c-induced oxidation of liposomes.

Two new protocols for exploring antioxidant-related chemical composition and reactivity are described: one based on a chronometric variation of a haemoglobin ascorbate peroxidase assay and one based on cytochrome c-induced oxidation of lecithin liposomes. Detailed accounts are given on their design, application, critical correlations with established methods and mechanisms. These assays are proposed to be physiologically relevant and bring new information regarding a real sample, both qualitative and quantitative. The well-known assays used for evaluation of antioxidant (re)activity are revisited and compared with these new methods. Extracts of the Hedera helix L. are examined as test case, with focus on seasonal variation and on leaf, fruit and flower with respect to chromatographic, spectroscopic and reactivity properties. According to the set of assays performed, winter are the most antioxidant, followed by summer leaves, and then by flowers and fruits.

Assessment of rosmarinic acid content in six Lamiaceae species extracts and their antioxidant and antimicrobial potential.

In the present study, six indigenous species of Lamiaceae family (Origanum vulgare L., Melissa officinalis L., Rosmarinus officinalis L., Ocimum basilicum L., Salvia officinalis L. and Hyssopus officinalis L.), have been analyzed to assess the rosmarinic acid, phenyl propane derivatives and polyphenolic contents and their antioxidant and antimicrobial potential. HPLC-MS method has been used for the analysis ofrosmarinicacid. The phenyl propane derivatives and total phenolic contents were determined using spectrophotometric method. The ethanolic extracts were screened for antioxidant activities by DPPH radical scavenging, HAPX (hemoglobin ascorbate per oxidase activity inhibition), and EPR (electron paramagnetic resonance) methods. The ethanolic extracts revealed the presence of rosmarinic acid in the largest amount in O. vulgare (12.40mg/g) and in the lowest in R. officinalis (1.33 mg/g). O. vulgare extracts exhibited the highest antioxidant capacity, in line with the rosmarinic acid and polyphenolic contents. The antimicrobial testing showed a significant activity against L. monocytogenes, S. aureus and C. albicans for all six extracts.

Antimicrobial and antioxidant activities and phenolic profile of Eucalyptus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill & L.A.S. Johnson leaves.

This study was performed to evaluate the in vitro antimicrobial and antioxidant activities and the phenolic profile of Eucalytus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill & L.A.S. Johnson leaves. Both leave extracts contain significant amounts of phenolic compounds, mainly flavonoids. Qualitative and quantitative analyses of the phenolic compounds were performed using a HPLC/MS method. The main flavonoid was hyperoside and its highest amount was found in E. globulus (666.42 ± 5.02 µg/g dw plant material). Regarding the flavonol profile, myricetin was the dominant compound and its highest amount was found in C. ficifolia leaves (124.46 ± 0.24 µg/g dw plant material). The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays, revealing an important antioxidant potential for both species. In the antimicrobial assays, C. ficifolia extract was found to be more active than E. globulus against both Gram-positive and Gram-negative bacterial strains with the exception of Bacillus subtilis. The results of the present study provide new valuable data regarding the bioactivities of these medicinal species.

Antioxidant activity evaluation involving hemoglobin-related free radical reactivity.

Two methods for the measurement of antioxidant capacity are described: one based on a chronometric variation of a hemoglobin ascorbate peroxidase assay and the other based on electron paramagnetic resonance (EPR) spectra collected upon alkaline treatment of ethanolic samples. The involved chemical mechanisms are discussed, alongside the most important benefits and shortcomings; the assays offer new qualitative and quantitative information on samples of biological as well as synthetic origin.

Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

Involvement of ferryl in the reaction between nitrite and the oxy forms of globins.

The reaction between nitrite and the oxy forms of globins has complex autocatalytic kinetics with several branching steps and evolves through chain reactions mediated by reactive species (including radicals) such as hydrogen peroxide, ferryl and nitrogen dioxide, starting with a lag phase, after which it proceeds onto an autocatalytic phase. Reported here are UV-Vis spectra collected upon stopped-flow mixing of myoglobin with a supraphysiological excess of nitrite. The best fit to the experimental data follows an A â†’ B â†’ C reaction scheme involving the formation of a short-lived intermediate identified as ferryl. This is consistent with a mechanism where nitrite binds to oxy myoglobin to generate an undetectable ferrous-peroxynitrate intermediate, whose decay leads to nitrate and ferryl. The ferryl is then reduced to met by the excess nitrite. DFT calculations reveal an essentially barrierless reaction between nitrite and the oxy heme, with a notable outer-sphere component; the resulting metastable ferrous-peroxynitrate adduct is found to feature a very low barrier towards nitrate liberation, with ferryl as a final product-in good agreement with experiment.

Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves.

This study was performed to evaluate the in vitro antioxidant and antimicrobial activities and the polyphenolic content of Lycium barbarum L. and L. chinense Mill. leaves. The different leave extracts contain important amounts of flavonoids (43.73 ± 1.43 and 61.65 ± 0.95 mg/g, respectively) and showed relevant antioxidant activity, as witnessed by the quoted methods. Qualitative and quantitative analyses of target phenolic compounds were achieved using a HPLC-UV-MS method. Rutin was the dominant flavonoid in both analysed species, the highest amount being registered for L. chinense. An important amount of chlorogenic acid was determined in L. chinense and L. barbarum extracts, being more than twice as high in L. chinense than in L. barbarum. Gentisic and caffeic acids were identified only in L. barbarum, whereas kaempferol was only detected in L. chinense. The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays revealing a better antioxidant activity for the L. chinense extract. Results obtained in the antimicrobial tests revealed that L. chinense extract was more active than L. barbarum against both Gram-positive and Gram-negative bacterial strains. The results suggest that these species are valuable sources of flavonoids with relevant antioxidant and antimicrobial activities.

Interactions between proteins and platinum-containing anti-cancer drugs.

Cisplatin and its congeners are well-known to exert their therapeutic effects on cancer via interaction with DNA in the cell nucleus. On the other hand, the undesirable side-effects of these drugs appear to also be linked, at least to some extent, to interaction of the platinum with proteins and peptides. For other classes of anticancer drugs, interaction with proteins is in fact the primary pathway whereby therapeutically-useful effects are achieved. Here, a review is given of the known instances of interaction of cisplatin and related compounds with proteins and biologically relevant peptides, with emphasis on structural and reactivity aspects.

Ascorbate peroxidase activity of cytochrome c.

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10-60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.