Radiolysis-Induced Oxidation of Bovine α-Crystallin

Abstract— Radiolysis of water by ionizing radiation results in the production of pure hydroxyl radicals. This technique, combined with analysis by tandem mass spectrometry (MS/MS), has been used to study the effect of hydroxyl radicals on the intact bovine α-crystallin protein. After exposure to -γ-irradiation, the oxidized α-crystallin was digested with trypsin and the resulting peptides were fractionated by reverse-phase HPLC. The isolated fractions were analyzed by matrix-assisted laser desorption ionization and by MS/MS to determine the locations and identities of the modifications. Structural analysis revealed that methionine 1 of αA- and αB-crystallin and methionine 68 of αB-crystallin were oxidized to methionine sulfoxide. Hydroxytryptophan was formed from each tryptophan residue in α-crystallin, although only tryptophan 9 of αA-crystallin was converted into N-for-mylkynurenine. This study has, for the first time, identified the sites of modification and the structures produced in the intact α-crystallin protein by exposure to hydroxyl radicals. By determining the consequences of in vitro exposure of α-crystallin to pure hydroxyl radicals, the in vivo contribution of this reactive oxygen species to the overall oxidative stress of the lens will be achieved from the identification of the modifications to α-crystallin purified from intact human lenses.     

Purification and Identification of Novel Myeloperoxidase Inhibitory Antioxidant Peptides from Tuna (Thunnas albacares) Protein Hydrolysates

Antioxidative peptides that inhibit myeloperoxidase (MPO) enzyme activity can effectively defend against oxidative stress damage. The antioxidant peptides from tuna protein were produced using alcalase hydrolysis and purified by ultrafiltration and Sephadex G-15, and the fractions with the highest free radicals scavenging ability and oxygen radical absorbance capacity (ORAC) values were sequenced using HPLC–MS/MS. Fifty-five peptide sequences were identified, 53 of which were successfully docked into MPO. The representative peptide ACGSDGK had better antioxidant activity and inhibition of MPO chlorination and peroxidation than the reference peptide hLF1-11. The docking model further showed intense molecular interactions between ACGSDGK and MPO, including hydrogen bonds, charge, and salt bridge interactions, which occluded the active site and blocked the catalytic activity of MPO. These results suggested that the antioxidant peptide ACGSDGK has the potential to inhibit oxidative stress and alleviate inflammation in vivo because of its inhibitory effect on the MPO enzyme.     

Protective effects of timosaponin BII on oxidative stress damage in PC12 cells based on metabolomics

Peroxide and oxygen free radicals are some of the causes of oxidative stress in brain tissue, and could lead to the change of brain structure and function. In addition, oxidative damage is one of the most important causes of the aging of the vast majority of tissues. The aim of this study is to investigate the protective effect of timosaponin BII on oxidative stress damage of PC12 induced by H₂O₂ using metabolomics based on the UHPLC‐Q‐TOF‐MS technique. Partial least‐squares discriminant analysis method was used to identify 35 metabolites as decisive marker compounds in a preliminary interpretation of the mechanism of the antioxidative effect of timosaponin BII. The majority of these metabolites are involved in the glutathione metabolism, amino acid metabolism, sphingolipid and glycerophospholipid metabolism. Our results suggest that timosaponin BII demonstrates systematic antioxidant effects in the PC12 oxidative damage cell model via the regulation of multiple metabolic pathways. These findings provide insight into the pathophysiological mechanisms underlying oxidative stress damage and suggest innovative and effective treatments for this disorder, providing a reliable basis for the development of novel therapeutic target in timosaponin BII treatment of oxidative stress.     

Chromatographic and electrophoretic methods for the analysis of biomarkers of oxidative damage to macromolecules (DNA, lipids, and proteins)

Free radicals and other reactive species can cause oxidative damage to biomolecules when oxidant species exceed the antioxidant defences in the body, resulting in oxidative stress. Oxidatively damaged products have been associated with aging as well as with the development of pathologies like cancer, cardiovascular disease, neurodegenerative disorders, diabetes, inflammation, etc. Reliable measurements of biomarkers of oxidative damage to macromolecules would afford information on the pre-disposition and prognosis of certain pathologies, being of utmost importance in evaluation of the effect of intervention with antioxidants on the incidence of diseases associated to oxidative stress. This review will present and compare different analytical methods, especially those involving chromatographic and electrophoretic techniques, commonly used for the analysis of biomarkers of oxidative damage to the three main macromolecules, namely oxidised DNA, lipid peroxidation products, and protein carbonyls.     

Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Antioxidant Activity of Acanthopanax senticosus Flavonoids in H2O2-Induced RAW 264.7 Cells and DSS-Induced Colitis in Mice

The redox reaction is a normal process of biological metabolism in the body that leads to the production of free radicals. Under conditions such as pathogenic infection, stress, and drug exposure, free radicals can exceed normal levels, causing protein denaturation, DNA damage, and the oxidation of the cell membrane, which, in turn, causes inflammation. Acanthopanax senticosus (A. senticosus) flavonoids are the main bioactive ingredients with antioxidant function. H₂O₂-treated RAW 264.7 cells and DSS-induced colitis in mice were used to evaluate the antioxidant properties of A. senticosus flavonoids. The results show that A. senticosus flavonoids can significantly downregulate the levels of ROS and MDA in H₂O₂-treated RAW 264.7 cells and increase the levels of CAT, SOD, and GPx. A. senticosus flavonoids can also increase the body weights of DSS-induced colitis mice, increase the DAI index, and ameliorate the shortening of the colon. ELISA experiments confirmed that A. senticosus flavonoids could reduce the level of MDA in the mouse serum and increase the levels of SOD, CAT, and GPx. Histopathology showed that the tissue pathological changes in the A. senticosus flavonoid group were significantly lower than those in the DSS group. The Western blot experiments showed that the antioxidant capacity of A. senticosus flavonoids was accomplished through the Nrf2 pathway. In conclusion, A. senticosus flavonoids can relieve oxidative stress in vivo and in vitro and protect cells or tissues from oxidative damage.     

Role of Melatonin in Angiotensin and Aging

The cellular utilization of oxygen leads to the generation of free radicals in organisms. The accumulation of these free radicals contributes significantly to aging and several age-related diseases. Angiotensin II can contribute to DNA damage through oxidative stress by activating the NAD(P)H oxidase pathway, which in turn results in the production of reactive oxygen species. This radical oxygen-containing molecule has been linked to aging and several age-related disorders, including renal damage. Considering the role of angiotensin in aging, melatonin might relieve angiotensin-II-induced stress by enhancing the mitochondrial calcium uptake 1 pathway, which is crucial in preventing the mitochondrial calcium overload that may trigger increased production of reactive oxygen species and oxidative stress. This review highlights the role and importance of melatonin together with angiotensin in aging and age-related diseases.     

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.     

Evaluation of UVA-Mediated Oxidative Damage to Proteins and Lipids in Extracorporeal Photoimmunotherapy

The combination of UVA and 8-methoxypsoralen (8-MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level < or = 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8-MOP concentrations resulted neither in an increase of malondialdehyde as a marker of lipid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co-trigger the photoimmunomodulatory effects of ECPI.     

聚酰胺热氧化降解机理

通过详细分析测定反应产物 ,研究了聚酰胺 (PA6)的热氧化降解机理 .用化学方法测定了PA6热氧化过程中几种主要基团含量的变化 ,对PA6热氧化的气相挥发产物进行收集和分析鉴定 ,还对经过氧化的PA6样品进行水解并用GC MS法对水解产物进行了分析鉴定 .在这些实验结果的基础上 ,提出了较完整的聚酰胺热氧化降解机理 .该机理包含了自由基在与酰胺基的羰基相连的亚甲基上的夺氢反应及一系列自由基加成环化和诱导断链的反应 ,能很好地解释反应产物的分析结果 .     

Identification and quantification of protein carbonylation using light and heavy isotope labeled Girard's P reagent

Protein carbonyls are one of the most widely studied markers of oxidative stress. Determining increases in the concentration of protein carbonyls known to be associated with neurodegenerative diseases, heart disease, cancer and ageing. Identification of carbonylation sites in oxidized proteins has been a challenge. Even though recent advances in proteomics has facilitate the identification of carbonylation sites in oxidized proteins, confident identification remains a challenge due to the complicated nature of oxidative damage and the wide range of oxidative modifications. Here, we report the development of a multiplexing strategy that facilitates confident carbonylated peptide identification through a combination of heavy and light isotope coding and a multi-step filtering process. This procedure involves (1) labeling aliquots of oxidized proteins with heavy and light forms of Girard's reagent P (GPR) and combining them in a 1:1 ratio along with (2) LC/MS and MALDI-MS/MS analysis. The filtering process uses LC/MS and MALDI-MS/MS data to rule out false positives by rejecting peptide doublets that do not appear with the correct concentration ratio, retention time, tag number, or resolution. This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in identification 13 distinct peptides. The competency of the method was validated in a complex mixture using oxidized transferrin in a yeast lysate as well as oxidized yeast. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. Analysis of yeast proteome stressed with hydrogen peroxide using this multiplexing strategy resulted in identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR.     

Electrochemical Study on DNA Damage Based on the Direct Oxidation of 8‐Hydroxydeoxyguanosine at an Electrochemically Modified Glassy Carbon Electrode

The study of DNA damage induced by Fenton reaction (Fe²⁺/H₂O₂) in vitro was performed based on the direct electrochemical oxidation of 8‐hydroxydeoxyguanosine (8‐OH‐dG), the biomarker of DNA oxidative damage, at an electrochemically modified glassy carbon electrode (GCE). The effects of antioxidants, such as ascorbic acid, and hydroxyl‐radical scavenger (mannitol) on the DNA damage were also investigated. 8‐OH‐dG, the oxidation product of guanine residues in DNA, has shown significantly oxidative peak on the electrochemically modified GCE. The oxidative peak current of 8‐OH‐dG was linear with the damaged DNA concentration in the range of 10–200 mg/L. The experimental results demonstrate that ascorbic acid has ambivalent effect on DNA oxidative stress. It can promote DNA oxidative damage when ascorbic acid concentration is below 1.5 mM and protect DNA from damage in the range of 1.5–2.5 mM. As a hydroxyl‐radical scavenger, mannitol inhibits significantly DNA oxidative damage. The influence of Fe²⁺, as reactant, and EDTA as iron chelator in the system were also studied. The proposed electrochemical method can be used for the estimation of DNA oxidative damage from new point of view.     

取代苯甲醛肟和取代苯乙酮肟电化学氧化机理的研究

研究了１０种取代苯甲醛肟和５种取代苯乙酮肟的电化学氧化机理。这二类肟的氧化电位随取代基吸电子能力的增大而增高，并与它们相应的Ｈａｍｍｅｔｔ常数有线性关系。它们在电化学氧化过程中均有ｉｍｉｎｏｘｙ自由基生成，但最后产物各不相同。用控制电位电解和紫外吸收光谱检法验证了部分电化学氧化产物。因此，电化学氧化法可作为由肟类产生ｉｍｉｎｏｘｙ自由基的一种方法。     

A mass spectrometry study of tirapazamine and its metabolites. insights into the mechanism of metabolic transformations and the characterization of reaction intermediates

Tandem mass spectrometry methods were used to study the sites of protonation and for identification of 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine), and its metabolites (3-amino-1,2,4-benzotriazine 1-oxide (3), 3-amino-1,2,4-benzotriazine 4-oxide (4), 3-amino-1,2,4-benzotriazine (5), and a related isomer 3-amino-1,2,4-benzotriazine 2-oxide (6). Fragmentation pathways of 3 and 5 indicated the 4-N-atom as the most likely site of protonation. Among the N-oxides studied, the 4-oxide (4) showed the highest degree of protonation at the oxygen atom. The differences in collision-induced dissociation of isomeric protonated 1-, 2- and 4-oxides allowed for their identification by LC/MS/MS. Gas phase and liquid phase protonation of tirapazamine occurred exclusively at the oxygen in the 4-position. A loss of OH radical from these ions (2(+)) resulted in ionized 3. Neutralization-reionization mass spectrometry (NR MS) experiments demonstrated the stability of the neutral analogue of protonated tirapazamine in the gas phase in the micro s time-frame. A significant portion of the neutral tirapazamine radicals (2) dissociated by loss of hydroxyl radical during the NR MS event, which indicates that previously proposed mechanisms for redox-activated DNA damage are reasonable. The activation energy for loss of hydroxyl radical from activated tirapazamine (2) was estimated to be approximately 14 kcal mol(-1). Stable neutral analogues of [3 + H](+) and [5 + H](+) ions were also generated in the course of NR MS experiments. Structures of these radicals were assigned to the molecules having an extra hydrogen atom at one of the ring N-atoms. Quantum chemical calculations of protonated 1, 3, 4 and 5 and the corresponding neutrals were performed to assist in the interpretation of experimental results and to help identify their structures.     

Electrospray tandem mass spectrometric analysis of zeaxanthin and its oxidation products

Carotenoids have been implicated in protection of the eye from light-mediated photo-toxicity caused by free radicals. Under conditions of normal oxidative stress the carotenoids serve as protective antioxidants; however, when the oxidative stress exceeds the antioxidant capacity, carotenoids can be oxidized into numerous cleavage products. The determination and identification of oxidized carotenoids in biological samples remains a major challenge due to the small sample size and low stability of these compounds. We investigated the reaction of various zeaxanthin cleavage products with O-ethyl hydroxylamine to evaluate their levels in a biological sample. For this, a sensitive and specific electrospray tandem mass spectrometry (ESI-MS/MS) was developed, avoiding the classical lower sensitive and specific HPLC-UV and fluorescence absorption methods. Protonated molecules [M + H](+) of carotenoids upon collision-induced dissociation produced a number of structurally characteristic product ions. A series of complicated clusters of product ions differing in 14 (CH(2))and 26 (C(2)H(2))Da was characteristic of the polyene chain of intact carotenoids. All carotenoid ethyl oximes of zeaxanthin cleavage products were characterized by the losses of 60 and 61 Da in their MS/MS spectra. Through the application of the LC/MS/MS method, we identified two oxime derivatives of 3-hydroxy-beta-ionone and 3-hydroxy-14'-apocarotenal with protonated molecules at m/z 252 and m/z 370 respectively, in a human eye sample.     

芯片毛细管电泳评估脂质体介导超氧化物歧化酶减低细胞内氧化应激

超氧化物歧化酶(SOD)可用作抗氧化的药物。它能催化并清除细胞内的活性氧组分(ROS),保护细胞免受自由基的氧化破坏。但是由于SOD分子量较大,难以透过细胞膜进入细胞内,显著降低了SOD的药效。本研究用激光共聚焦荧光显微镜拍摄的荧光图像说明,纳米脂质体可介导SOD进入细胞。用芯片毛细管电泳激光诱导荧光分析法(MCE-LIF)测定单细胞中ROS和谷胱甘肽(GSH)的荧光信号强度,评估了用脂质体包裹的SOD与细胞作用的抗氧化效果。用脂质体包裹的SOD与肝癌细胞共培养2h,与直接用SOD作用于肝癌细胞相比较,细胞内ROS明显降低,GSH明显提高。实验结果说明,用脂质体包裹SOD是一种减低细胞内氧化应激的有效给药途径。     

Microorganisms: A Potential Source of Bioactive Molecules for Antioxidant Applications

Oxidative stress originates from an elevated intracellular level of free oxygen radicals that cause lipid peroxidation, protein denaturation, DNA hydroxylation, and apoptosis, ultimately impairing cell viability. Antioxidants scavenge free radicals and reduce oxidative stress, which further helps to prevent cellular damage. Medicinal plants, fruits, and spices are the primary sources of antioxidants from time immemorial. In contrast to plants, microorganisms can be used as a source of antioxidants with the advantage of fast growth under controlled conditions. Further, microbe-based antioxidants are nontoxic, noncarcinogenic, and biodegradable as compared to synthetic antioxidants. The present review aims to summarize the current state of the research on the antioxidant activity of microorganisms including actinomycetes, bacteria, fungi, protozoa, microalgae, and yeast, which produce a variety of antioxidant compounds, i.e., carotenoids, polyphenols, vitamins, and sterol, etc. Special emphasis is given to the mechanisms and signaling pathways followed by antioxidants to scavenge Reactive Oxygen Species (ROS), especially for those antioxidant compounds that have been scarcely investigated so far.     

低能氮离子诱发丙酮与重水溶液的反应机理

利用氮气火花放电产生离子,其中的正离子在阴极位降的加速下“注入”到丙酮的重水溶液中,诱发其中的化学反应.利用气相色谱 质谱（GC MS）分析离子注入后的样品,证实有氘代产物（CH3COCH2D）、氘羟基取代产物（CH3COCH2OD）生成,这表明低能N＋诱发重水溶液中的反应主要是由于重水分子分解产生的自由基引起的,其中氘自由基和氘羟基自由基起重要作用;同时,产物中还检测到氘代乙酸（CH3COOD）和氘氨基丙酮（CH3COCH2ND2）,说明反应是在氧化性氛围中进行的,氮离子俘获重水中的氘形成氘氨基可能是氘氨基取代产物生成的主要原因,也是氮“沉积”在溶液中的重要形式.这些结果对初步揭示低能离子诱发水溶液的反应机理具有一定的意义.     

Free Radicals as a Double-Edged Sword: The Cancer Preventive and Therapeutic Roles of Curcumin

Free radicals, generally composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated in the body by various endogenous and exogenous systems. The overproduction of free radicals is known to cause several chronic diseases including cancer. However, increased production of free radicals by chemotherapeutic drugs is also associated with apoptosis in cancer cells, indicating the dual nature of free radicals. Among various natural compounds, curcumin manifests as an antioxidant in normal cells that helps in the prevention of carcinogenesis. It also acts as a prooxidant in cancer cells and is associated with inducing apoptosis. Curcumin quenches free radicals, induces antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and upregulates antioxidative protein markers–Nrf2 and HO-1 that lead to the suppression of cellular oxidative stress. In cancer cells, curcumin aggressively increases ROS that results in DNA damage and subsequently cancer cell death. It also sensitizes drug-resistant cancer cells and increases the anticancer effects of chemotherapeutic drugs. Thus, curcumin shows beneficial effects in prevention, treatment and chemosensitization of cancer cells. In this review, we will discuss the dual role of free radicals as well as the chemopreventive and chemotherapeutic effects of curcumin and its analogues against cancer.     

Novel biomarkers of protein oxidation sites and degrees using horse cytochrome c as the target by mass spectrometry

Biomarkers held both incredible application and significant challenge in probing the oxidation mechanisms of proteins under oxidative stress. Here, mass spectrometry (MS) coupled with liquid chromatography (LC) was applied to establish a new pipeline to probe the oxidation sites and degrees of horse cytochrome c (HCC) with its oxidative products serving as the biomarkers. Samples of native and UV/H(2)O(2) oxidized HCCs were digested by trypsin and subjected to biomarker discovery using LC/MS and tandem mass spectrometry (MS/MS). Experiment results proved that the main oxidation sites were located at Cys(14), Cys(17), Met(65) and Met(80) residues in peptides C(14)AQC(heme)HTVEK(22), C(14)AQCHTVEK(22), E(60)ETLMEYLENPKK(73), M(80)IFAGIK(86) and M(80)IFAGIKK(87). Quantitative analysis on the oxidized peptides showed the oxidation degrees of target sites had positive correlations with extended oxidation dose and controlled by residues types and their accessibility to solvent molecules. Being able to provide plentiful information for the oxidation sites and oxidation degrees, the identified oxidized products were feasibility biomarkers for HCC oxidation, compared with the conventional protein carbonyl assay.