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1.
Molecules ; 26(21)2021 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-34771139

RESUMO

Herein, a method based on selective piazselenol formation is applied for total selenium determination in biofortified Allium species. Piazselenol is formed by reacting Se(IV) with an aromatic diamine, namely 4-nitro-1,2-phenylenediamine, in acidic medium. Samples were digested in a nitric acid/hydrogen peroxide open system, followed by selenate reduction in hydrochloric acid. Reaction conditions were optimized in terms of pH, temperature, reaction time, and other auxiliary reagents for interference removal, namely, EDTA and hydroxylamine. For the extraction of the selectively formed 4-nitro-piazselenol, micro-solid-phase extraction (µSPE) was applied, and the analysis and detection of the corresponding complex was performed by HPLC coupled with DAD. An external standard calibration curve was developed (R2 = 0.9994) with good sensitivity, and was used to calculate the total selenium content from several Allium plants material, with good intermediate precision (RSD% < 16%). The accuracy of the method was evaluated using both, a comparison with an accepted reference method from our previously published data, as well as three certified reference material with recoveries between 84-126%. The limit of detection was determined to be 0.35 µg/g (in solids) and 1.1 µg/L (in solution), while the limit of quantification was 1.07 µg/g and 3.4 µg/L (in solution). Using the proposed method, selenium content can be quickly and accurately determined in several types of samples. In addition, this study present experimental conditions for overcoming the interferences that might be encountered in selenium determination using piazselenol.

2.
Anal Methods ; 12(25): 3260-3267, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32930189

RESUMO

Advanced chemometric methods, such as fuzzy c-means (FCM), a fuzzy divisive hierarchical clustering algorithm (FDHC), and fuzzy divisive hierarchical associative-clustering (FDHAC), which offer the excellent possibility to associate each fuzzy partition of samples with a fuzzy set of characteristics (features), have been successfully applied in this study. FDHAC, a method that utilizes specific regions of chromatographic fingerprints or specific peaks as a fuzzy set of characteristics, was effectively applied to the characterization and classification of medicinal plant extracts according to their antioxidant capacities, using their chromatographic profiles monitored at 242, 260, 280, 320, 340, and 380 nm via HPLC with a multistep isocratic and gradient elution system and diode array detection (HPLC-DAD). What is quite new is the partitioning of the chromatographic retention time ranges and peaks (markers) and their association with different plant extract samples with high, moderate or low antioxidant capacity. Furthermore, the degrees of membership of fingerprints (fuzzy markers) are highly relevant with respect to the (dis)similarity of samples because they indicate both the positions and degrees of association of chromatographic peaks from different classes or individual samples. The obtained results clearly demonstrate the efficiency and information power of these advanced fuzzy methods for medicinal plant characterization and authentication, and this study generates the premise for a new chemometrics approach with high-impact for use in analytical chemistry and other fields.


Assuntos
Extratos Vegetais , Plantas Medicinais , Algoritmos , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados
3.
Pharmaceuticals (Basel) ; 13(6)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471171

RESUMO

Excess ascorbate (as expected in intravenous treatment proposed for COVID-19 management, for example) oxidizes and/or degrades hemoglobin and albumin, as evidenced by UV-vis spectroscopy, gel electrophoresis, and mass spectrometry. It also degrades hemoglobin in intact blood or in isolated erythrocytes. The survival rates and metabolic activities of several leukocyte subsets implicated in the antiviral cellular immune response are also affected. Excess ascorbate is thus an unselective biological stress agent.

4.
Food Funct ; 11(6): 5293-5307, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32458896

RESUMO

Quercetin, one of the most abundant flavonoids in plant-based foods, commonly occurs in nature in various glycosylated forms. There is still a less explored aspect regarding the cause of diversity of its glycosides, depending on the sugar moiety attached. This work focuses on four wide-spread quercetin glycosides-hyperoside, isoquercitrin, quercitrin and rutin-by testing the property-tuning capacity of different sugar moieties and thus explains and predicts some of their functions in plant-based foods. The electron paramagnetic spectra of the semiquinone anion radicals of these glycosides were interpreted in terms of hyperfine coupling constants and linewidths, highlighting a clear link between spin density trends, the identity of the bound sugar, and their reactivity corroborated with their modelled structures. Redox potential and lipophilicity were connected to a specific flavonoid-enzyme interaction and correlated with their prooxidant reactivity assessed by oxidation of ferrous hemoglobin. Hyperoside and isoquercitin-galactose and glucose glycosides-exhibit the highest prooxidant reactivity owing to their lowest redox potential and lipophilicity whereas rutin and quercitrin-rutinose and rhamnose glycosides-behave vice versa. The ability of the tested glycosides to undergo HAT or SET-type reactions has also been tested using five different analytical assays, including inhibition of cytochrome c-triggered liposome peroxidation. In most cases, rutin proved to be the most unreactive of the four tested glycosides considering either steric or redox reasons whereas the reactivity hierarchy of the other three glycosides were rather assay dependent.


Assuntos
Glicosídeos/química , Quercetina/análogos & derivados , Rutina/química , Antioxidantes/química , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Sequestradores de Radicais Livres/química , Hidrogênio/química , Lipídeos/química , Oxidantes/química , Oxirredução , Quercetina/química , Rutina/farmacologia
5.
Bioelectromagnetics ; 41(3): 200-212, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32030775

RESUMO

The morphophysiological response of Phaseolus vulgaris L. to low-power electromagnetic radiation was investigated in order to assess the potential harmful effects of long-term continuous exposure. The plants were grown in two separate electromagnetic field (EMF) shielded rooms, in a controlled, greenhouse-like environment. One batch was continuously irradiated during the growth period (from sowing to maturity) and the other one was used as a reference. An unmodulated signal at 915 MHz (the central frequency between the uplink and downlink of the GSM900 mobile communications band) was used, with a maximum power density of 10 mW/m2 measured near the plants. The plants were analyzed using ultraviolet-visible, statistical, morphometric, and electron microscopy methods. Significant differences were observed regarding the height of the plants, number of inflorescences, and chlorophyll and carotenoid content, all closely connected with the ultrastructural changes observed in the leaves. The irradiated batch grew higher (19% increase in plant height, 20% increase in stem and leaves' dry mass), with 18% fewer inflorescences, and extremely long roots (34% increase in dry mass). The ultrastructure of the irradiated leaves showed irregular cells and a higher content of plastoglobules in the chloroplasts. All results indicate that the irradiated plants suffered significant morphological modifications during their long-term exposure to the specific EM radiation. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.


Assuntos
Clorofila , Campos Eletromagnéticos , Phaseolus/fisiologia , Folhas de Planta , Carotenoides/análise , Carotenoides/metabolismo , Clorofila/análise , Clorofila/metabolismo , Inflorescência , Phaseolus/química , Folhas de Planta/química , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Ondas Ultrassônicas
6.
PLoS One ; 15(1): e0225530, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31961889

RESUMO

Yellow laccases lack the typical blue type 1 Cu absorption band around 600 nm; however, multi-copper oxidases with laccase properties have been reported. We provide the first evidence that the yellow laccase isolated from Sclerotinia sclerotiorum is obtained from a blue form by covalent, but nevertheless reversible modification with a phenolic product. After separating the phenolics from the extracellular medium, a typical blue laccase is obtained. With ABTS as model substrate for this blue enzyme, a non-natural purple adduct is formed with a spectrum nearly identical to that of the 1:1 adduct of an ABTS radical and Tyr. This modification significantly increases the stability and substrate affinity of the enzyme, not by acting primarily as bound mediator, but by structural changes that also alters the type 1 Cu site. The HPLC-MS analyses of the ABTS adduct trypsin digests revealed a distinct tyrosine within a unique loop as site involved in the modification of the blue laccase form. Thus, S. sclerotiorum yellow laccase seems to be an intrinsically blue multi-copper oxidase that boosts its activity and stability with a radical-forming aromatic substrate. This particular case could, at least in part, explain the enigma of the yellow laccases.


Assuntos
Ascomicetos/enzimologia , Lacase/metabolismo , Tirosina/metabolismo , Biocatálise , Cor , Concentração de Íons de Hidrogênio , Fenóis/metabolismo , Ligação Proteica
7.
Free Radic Biol Med ; 146: 189-197, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31705959

RESUMO

Superoxide radical is one of the main players when it comes to oxidative stress. Even if in itself is moderately reactive and can cause the degradation of very few biologically relevant macromolecules, it can dismutate to hydrogen peroxide followed by a possible conversion to hydroxyl radical. In order to protect the internal environment against reactive oxygen species, plants have evolved a line of defence made from secondary metabolites with versatile redox properties, such as flavonoids and phenolic acids. Their characteristics are highly modulated by pH, as they turn into prooxidant compounds as it increases. Reported here are the behaviour and clear patterns in reactivity towards superoxide anion radical of four classes of plant phenolics as a pH function. The reactivity towards superoxide radical in acidic conditions has been studied by use of oscillating Briggs-Rauscher reaction with a new spectroelectrochemical experimental setup, by recording the absorbance in high quality for the first time. Some mechanistic intricacies have also been explored with regard to this method. Reactivity modulation at neutral and slightly basic pH has been assayed by superoxide radical scavenging ability using nitroblue tetrazolium as a substrate. For stronger alkaline pHs studies, Electron Paramagnetic Resonance was exploited. Hydroxybenzoic acids tend to be the least reactive species at all tested pH values. Hydroxycinnamic acids have their activity towards superoxide radical decreased as the pH increases, whereas flavonoids act vice versa.


Assuntos
Polifenóis , Superóxidos , Sequestradores de Radicais Livres , Radical Hidroxila , Fenóis , Espécies Reativas de Oxigênio
8.
BMC Complement Altern Med ; 19(1): 289, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664997

RESUMO

BACKGROUND: Natural extracts with beneficial biological activities are nowadays of high interest, in various treatment or prophylaxis. Hypericum capitatum has been known for its curative effects for centuries and its extracts have become of interest due to their distinct activity among other Hypericaceae members. In this study, further light is aimed to be shed on the secondary-metabolites composition of H. capitatum extracts, using chromatographic techniques and Electron paramagnetic resonance profiles in alkaline medium. Considering that no previous works explored the anti-inflammatory activity of H. capitatum, here, an in vivo study is also designed in order to evaluate this property by assessing the impact of one of H. capitatum extracts in ameliorating turpentine oil-induced inflammation on rats and to quantify their blood antioxidants level. METHODS: Chromatographic techniques and Electron paramagnetic resonance spectroscopy were used in order to describe the chemical profile in different parts of the plant. The in vivo study on turpentine-oil induced inflammation in rats included three doses of H. capitatum extract expressed in rutin concentration. Oxidative stress was measured using total oxidative status, total antioxidant capacity, oxidative stress index, 3-nitrotyrosine, nitric oxide, malondialdehyde, superoxide dismutase, catalase and the inflammatory response was evaluated by performing a complete blood cells count and C reactive protein. RESULTS: The extract was remarkably rich in rutin; however, other polyphenolic-like minor components appeared important in explaining the observed biological properties. The tested extract prevents the increase of inflammation-induced white blood cell count, number of neutrophils, and serum nitric oxide, and did so in a dose-dependent manner, similarly to the positive control-diclofenac. In addition, the same extract appeared to be a good alternative to diclofenac to restore total oxidative status, thiobarbituric active reactive species, total proteins and C reactive proteins. Moreover, antioxidant enzymes such as catalase, superoxide dismutase and total serum thiol concentration were significantly increased by the tested extract. CONCLUSIONS: Due to its powerful reservoir rich in rutin, H. capitatum extract depicted its in vivo antioxidant and anti-inflammatory effects indicating it to be a good alternative to conventional drugs for oxidative stress protection.


Assuntos
Anti-Inflamatórios/administração & dosagem , Hypericum/química , Inflamação/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Rutina/administração & dosagem , Animais , Anti-Inflamatórios/química , Catalase/metabolismo , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/química , Ratos , Ratos Wistar , Rutina/análise , Superóxido Dismutase/metabolismo , Terebintina/efeitos adversos
9.
Free Radic Biol Med ; 137: 110-115, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31035002

RESUMO

Hemoglobin in its ferryl form oxidizes hydrogen sulfide and is transformed to sulfhemoglobin, where the sulfur is inserted covalently at the heme edge. Shown here is evidence that-as previously proposed by others-this process involves oxidation of hydrogen sulfide to a sulfanyl radical detectable by spin-trapping in electron paramagnetic resonance (EPR) spectroscopy. The yields and rates of formation of sulfhemoglobin as well as of the sulfanyl radical are affected by the same factors that affect the reactivity of hemoglobin ferryl, in bovine hemoglobin and in phytoglobins as well. A freely-diffusing sulfanyl radical is thus proposed to be involved in sulfhemoglobin formation. Catalase is shown to accelerate this process due to a previously described hydrogen sulfide oxidase activity, within which EPR evidence for sulfanyl generation is shown here for the first time. The reaction of preformed ferryl with hydrogen sulfide-in absence of hydrogen peroxide-is studied by stopped-flow at several pH values and explained in light of reactivity and redox potential control.


Assuntos
Heme/metabolismo , Hemoglobinas/metabolismo , Sulfa-Hemoglobina/metabolismo , Animais , Catalase/metabolismo , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Hemoglobinas/química , Peróxido de Hidrogênio/química , Sulfeto de Hidrogênio/química , Concentração de Íons de Hidrogênio , Oxirredução , Sulfa-Hemoglobina/química , Compostos de Sulfidrila
10.
Sci Rep ; 8(1): 13714, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209406

RESUMO

Despite a recent increase in interest towards phytoglobins and their importance in plants, much is still unknown regarding their biochemical/biophysical properties and physiological roles. The present study presents data on three recombinant Arabidopsis phytoglobins in terms of their UV-vis and Raman spectroscopic characteristics, redox state control, redox potentials and autoxidation rates. The latter are strongly influenced by pH for all three hemoglobins - (with a fundamental involvement of the distal histidine), as well as by added anion concentrations - suggesting either a process dominated by nucleophilic displacement of superoxide for AtHb2 or an inhibitory effect for AtHb1 and AtHb3. Reducing agents, such as ascorbate and glutathione, are found to either enhance- (presumably via direct electron transfer or via allosteric regulation) or prevent autoxidation. HbFe3+ reduction was possible in the presence of high (presumably not physiologically relevant) concentrations of NADH, glutathione and ascorbate, with differing behaviors for the three globins. The iron coordination sphere is found to affect the autoxidation, redox state interconversion and redox potentials in these three phytoglobins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Hemoglobinas/metabolismo , Ácido Ascórbico/metabolismo , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , NAD/metabolismo , Oxirredução , Superóxidos/metabolismo
11.
PLoS One ; 13(7): e0200022, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29969484

RESUMO

Galium verum is a well-known medicinal plant which is used in various pathologies. G. verum extracts are characterized here using chromatography, where among the rich pool of phenolic acids of flavonoids two known anti-stress modulators, chlorogenic acid and rutin are identified in high quantities. Additionally, the extracts are characterized using a series of in vitro assays (EPR, DPPH, TPC and TEAC). Considering the chemical findings, the potential beneficial effects of the G. verum extract are explored here in a living organism exposed to stress induced oxidative damages. Thus, the biochemical-modulatory and antioxidant roles of two doses of G. verum extract are examined in animals exposed to acute restraint and dark stress (S). The animals were divided in groups [control, S, SG1 (exposed to 25 mg G. verum extract), SG2 (50 mg extract)]. Increased levels of lipid peroxidation (TBARS from 4.43 to 8.06 nmol/mL), corticosterone from 0.43 to 1.96 µg/dL and epinephrine from 44.43 to 126.7 µg/mL, as well as decreased antioxidant enzymes activities (SOD/CAT) were observed in the S group. The G. verum extract afforded a near-normal equilibrium within the biochemical parameters of animals exposed to RS, by reducing oxidative damage (TBARS at a 3.73 nmol/mL; CS at 0.90 µg/dL; EP at 63.72 µg/mL) and by restoring the antioxidant balance.


Assuntos
Antioxidantes/farmacologia , Escuridão/efeitos adversos , Galium/química , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Restrição Física/psicologia , Estresse Psicológico/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Colesterol/metabolismo , Corticosterona/sangue , Relação Dose-Resposta a Droga , Epinefrina/sangue , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Polifenóis/farmacologia , Ratos , Ratos Wistar , Estresse Psicológico/sangue , Estresse Psicológico/enzimologia , Estresse Psicológico/etiologia
12.
Free Radic Biol Med ; 124: 260-274, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-29928975

RESUMO

Hemoglobin has previously been shown to display ascorbate peroxidase and urate peroxidase activity, with measurable Michaelis-Menten parameters that reveal a particularly low Km for ascorbate as well as for urate - lower than the respective in vivo concentrations of these antioxidants in blood. Also, direct detection of a hemoglobin-ascorbate interaction was possible by monitoring the 1H-NMR spectrum of ascorbate in the presence of hemoglobin. The relative difference in structures between ascorbate and urate may raise the question as to exactly what the defining structural features would be, for a substrate that binds to hemoglobin with high affinity. Reported here are Michaelis-Menten parameters for hemoglobin acting as peroxidase against a number of other substrates of varying structures - gallate, caffeate, rutin, 3-hydroxyflavone, 3,6-dihydroxyflavone, quercetin, epicatechin, luteolin - all with high affinities (some higher than those of physiologically-relevant redox partners of Hb - ascorbate and urate). Moreover, this high affinity appears general to animal hemoglobins. 1H-NMR and 13C-NMR spectra reveal a general pattern wherein small hydrophilic antioxidants appear to all have their signals affected, presumably due to binding to hemoglobin. Fluorescence and calorimetry measurements confirm these conclusions. Docking calculations confirm the existence of binding sites on hemoglobin and on myoglobin for ascorbate as well as for other antioxidants. Support is found for involvement of Tyr42 in binding of three out of the four substrates investigated in the case of hemoglobin (including ascorbate and urate, as blood-contained relevant substrates), but also for Tyr145 (with urate and caffeate) and Tyr35 (with gallate).


Assuntos
Antioxidantes/química , Antioxidantes/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Animais , Bovinos , Simulação de Acoplamento Molecular , Oxirredução
13.
New Phytol ; 217(2): 613-624, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28277608

RESUMO

The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N-end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time. We gained mechanistic insights into PRT1 substrate preference and activation by monitoring live ubiquitination using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization. The enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc instantaneously and with significantly reduced reagent consumption. We demonstrated that PRT1 is indeed an E3 ligase, which has been hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.


Assuntos
Arabidopsis/metabolismo , Sistemas Computacionais , Corantes Fluorescentes/metabolismo , Ubiquitinação , Proteínas de Arabidopsis/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Proteólise , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
14.
J Inorg Biochem ; 179: 32-39, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29156293

RESUMO

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.


Assuntos
Hemoglobinas/química , Sulfeto de Hidrogênio/química , Ferro/química , Mioglobina/química , Animais , Bovinos , Cavalos , Concentração de Íons de Hidrogênio , Cinética , Miocárdio/química , Oxirredução
15.
Methods Mol Biol ; 1450: 55-83, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27424746

RESUMO

In order to determine the stability of a protein or protein fragment dependent on its N-terminal amino acid, and therefore relate its half-life to the N-end rule pathway of targeted protein degradation (NERD), non-Methionine (Met) amino acids need to be exposed at their amino terminal in most cases. Per definition, at this position, destabilizing residues are generally unlikely to occur without further posttranslational modification of immature (pre-)proproteins. Moreover, almost exclusively, stabilizing, or not per se destabilizing residues are N-terminally exposed upon Met excision by Met aminopeptidases. To date, there exist two prominent protocols to study the impact of destabilizing residues at the N-terminal of a given protein by selectively exposing the amino acid residue to be tested. Such proteins can be used to study NERD substrate candidates and analyze NERD enzymatic components. Namely, the well-established ubiquitin fusion technique (UFT) is used in vivo or in cell-free transcription/translation systems in vitro to produce a desired N-terminal residue in a protein of interest, whereas the proteolytic cleavage of recombinant fusion proteins by tobacco etch virus (TEV) protease is used in vitro to purify proteins with distinct N-termini. Here, we discuss how to accomplish in vivo and in vitro expression and modification of NERD substrate proteins that may be used as stability tester or activity reporter proteins and to characterize potential NERD substrates.The methods to generate artificial substrates via UFT or TEV cleavage are described here and can be used either in vivo in the context of stably transformed plants and cell culture expressing chimeric constructs or in vitro in cell-free systems such as rabbit reticulocyte lysate as well as after expression and purification of recombinant proteins from various hosts.


Assuntos
Biologia Molecular/métodos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Ubiquitina/química , Sequência de Aminoácidos/genética , Animais , Sistema Livre de Células , Metionina/química , Proteólise , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/genética , Reticulócitos/química , Especificidade por Substrato , Ubiquitina/genética
16.
Nat Prod Res ; 30(11): 1315-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26208459

RESUMO

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.


Assuntos
Antioxidantes/farmacologia , Lipossomos/química , Peroxidases/metabolismo , Animais , Antioxidantes/química , Bioensaio/métodos , Citocromos c/farmacologia , Flores/química , Frutas/química , Hemoglobinas , Humanos , Oxirredução/efeitos dos fármacos , Extratos Vegetais/química , Folhas de Planta/química , Estações do Ano
17.
Methods Mol Biol ; 1208: 247-55, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25323512

RESUMO

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.


Assuntos
Antioxidantes/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Radicais Livres/metabolismo , Hemoglobinas/metabolismo , Animais , Ascorbato Peroxidases/metabolismo , Ácido Ascórbico/metabolismo , Bovinos , Oxirredução , Fatores de Tempo
18.
Molecules ; 19(5): 5490-507, 2014 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-24786688

RESUMO

This study was designed to examine the in vitro antioxidant and antimicrobial activities and to characterize the polyphenolic composition of the ethanolic extracts of Hyssopus officinalis, Ocimum basilicum and Teucrium chamaedrys. Qualitative and quantitative analysis of the major phenolic compounds were conducted using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The total polyphenols, caffeic acid derivatives and flavonoids content was spectrophotometrically determined. The phenolic profile showed the presence of phenolic acid derivatives (caftaric, gentisic, caffeic, p-coumaric, chlorogenic and ferulic acids), flavonoid glycosides (rutin, isoquercitrin and quercitrin) and free flavonoid aglycons (luteolin, quercetin), in different concentrations. DPPH radical scavenging assay, Trolox equivalent antioxidant capacity (TEAC) method, hemoglobin ascorbate peroxidase activity inhibition (HAPX) assay, and electron paramagnetic resonance (EPR) radicals detection were employed, revealing several aspects of the antioxidant activities of these species. The antimicrobial tests were performed using the disk diffusion assay. These extracts contained a large amount of the polyphenolic compounds (77.72, 175.57, and 243.65 mg/g, respectively), and they showed a good antioxidant activity, as witnessed by a number of methods. T. chamaedrys had a high antimicrobial activity. Besides their antioxidant activity, the antimicrobial effect of these extracts confirms the biological activities of these herbal medicinal products.


Assuntos
Anti-Infecciosos/farmacologia , Antioxidantes/isolamento & purificação , Bactérias/efeitos dos fármacos , Fenóis/isolamento & purificação , Anti-Infecciosos/isolamento & purificação , Antioxidantes/química , Antioxidantes/farmacologia , Lamiaceae/química , Ocimum basilicum/química , Fenóis/farmacologia , Extratos Vegetais , Teucrium/química
19.
Biomacromolecules ; 15(5): 1920-7, 2014 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-24716617

RESUMO

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.


Assuntos
Substitutos Sanguíneos/metabolismo , Hemeritrina/metabolismo , Hemoglobinas/metabolismo , Oxigênio/metabolismo , Peroxidase/metabolismo , Substitutos Sanguíneos/química , Hemeritrina/química , Hemoglobinas/química , Células Endoteliais da Veia Umbilical Humana/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Oxirredução , Oxigênio/química , Peroxidase/química
20.
Pharmaceuticals (Basel) ; 6(7): 867-80, 2013 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24276319

RESUMO

Reviewed here are recent attempts to produce protein-based artificial oxygen carriers ("blood substitutes"). Most of these involve chemical or physical modifications on hemoglobin, although a recent line of research using hemerythrin instead of hemoglobin is also described. The focus is set on the extent to which these modifications alter the redox reactivity of the proteins, and on ways in which this can be done systematically and purposefully, within the framework of a working hypothesis where redox side-reactions hold an important role in the physiological outcome of experimental transfusions with artificial oxygen carriers.

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