新型兼具GPX,SOD, CAT活性的水杨醛Schiff碱衍生物的合成

以5-磺基水杨醛为母体, 经氯甲基化和硒化反应引入催化基团-SeH, 此化合物经空气氧化、Schiff碱反应以及锰螯合反应, 最终得到一种新型的水杨醛Schiff碱类谷胱甘肽过氧化物酶(GPX)模拟物. 测定了该模拟物的红外光谱、核磁共振谱、质谱和抗氧化能力. 此模拟物同时兼具超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性, 水溶性好, 保护线粒体免遭氧化损伤能力强, 具有作为药物前体的潜力.     

超氧化物歧化酶化学模拟的新进展

超氧化物歧化酶在生物体内特异性地催化超氧离子自由基（O2˙ˉ）的歧化反应, 具有抗氧化、抗癌症、抗炎症等重要生理学作用。近年来,超氧化物歧化酶的化学模拟倍受关注并引发人们极广泛的研究兴趣。本文系统综述了超氧化物歧化酶模拟物在设计合成及应用上的新近研究进展。     

Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species

In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O₂⁻, hydroxyl radical: OH, hydrogen peroxide: H₂O₂, hypochlorite anion: ClO⁻, singlet oxygen: ¹O₂, and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O₂⁻ while one CL reagent selectively detected OH. However, no CL reagent had selectivity on the detection of H₂O₂, ClO⁻, ¹O₂ and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS.As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O₂⁻, and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL⁻¹, respectively. Significant correlation (r = 0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.     

Biological Activity of Flavonoids Copper Complexes

Three flavonoid copper(II) complexes Cu₂(quercetin)(CH₃COO)₃(CH₃OH) ( 1 ), Cu(anthrarufin)(CH₃COO)·1/2H₂O ( 2 ) and Cu(naringin)(OCH₃)(CH₃OH)₂ ( 3 ) have been synthesized and characterized by elemental analysis, IR, electronic absorption and EPR (X‐band) spectroscopy. The complexes have a strong protective action over the Δsod1 mutant of S. cerevisiae against reactive oxygen radicals generated by an external source of free radicals (H₂O₂ or the superoxide‐generating, menadione). On the other hand, the complexes cleave DNA efficiently even in the absence of reducing agents. The main reactive oxygen species responsible for the DNA strand cleavage have been determined using radical scavengers. A probably mechanism of the DNA damage is proposed.     

A mini-review and perspective on ferroptosis-inducing strategies in cancer therapy

This review summarized recent advances and challenges in ferroptosis-based anticancer strategies through Fenton reaction or GPX4 inactivation, with highlights on nanomaterials and perspectives on future development of next-generation ferroptosis-inducing agents based on diverse polyvalent metals.     

具有cGPX活力的含硒抗体酶的酶学及动力学性质研究

通过单克隆抗体制备技术得到三株特异结合半抗原4(GSH-S-DNP二苄酯)的单克隆抗体HB4,HB5和HB7.抗体经两步化学诱变得到具有细胞谷胱甘肽过氧化物酶(cGPX)活性的含硒抗体酶mHB4,mHB5和mHB7,活力分别为170,1867,32U/μmol.其中mHB5的活力是天然兔肝cGPX的0.32倍,m4A4的1.51倍.等离子体-质谱(ICP/MS)测得每分子含硒抗体酶分子中大约存在2个硒原子.mHB5的最适pH为8.6～8.8.在pH值范围为7.0和37℃条件下,mHB5催化GSH和H₂O₂或t-ROOH反应的二级速率常数为:_k+1(H₂O₂)9.71×10⁶L/(mol·min),_k+1(t-ROOH)5.99×10⁵L/(mol·min).mHB5使非酶催化反应速率提高了9.8×10⁶和3.7×10⁵倍.     

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.     

Purification and Characterization of Superoxide Dismutase （SOD） from Camellia Pollen

A superoxide dismutase（ SOD ） was purified to homogeneity from fresh camellia pollen by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose（ DE52 ）, Sephadex G-100 and phenyl sepharose^TM 6 Fast Flow columns. Its specific activity could reach to 4034 U/mg protein and it was determined to be Cu/ Zn-SOD according to its different sensitivities to different inhibitors. The molecular weight of the SOD and its subunit were 69500 and 34700, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）, which implicates that the SOD in camellia pollen is a dimmer composed of two identical subunits. The isoelectric point of the enzyme was determined to be 4. 1 by isoelectric focusing electrophoresis and the N-terminal amino acid was identified to be Gly by the DNS-Cl method. Its α-Helix was also calculated to be approximately 21.8% according to the circular dichroism（CD） spectra.     

Purification and Properties of Superoxide Dismutase（SOD） in Allium Sativum

Superoxide dismutases(SODs) were purified to homogeneity from Allium Sativum by means of ammoni-um sulfate precipitation and column chromatography with DEAE--cellulose (DE52) and Sephadex G-75. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-AGE), Allium Sativum is predicted to contain four SODs. The molecular weights of the native SODs are 41.3 kD, 37. 0 kD, 35.2 kD and 31.0 kD, which consist of subunits of 20. 7 kD, 18. 4 kD, 17. 7 kD and 15.4 kD respectively. Because of their specific sensitivity to hydrogen peroxide, cyanogens potassium and chloroform-alcohol, the SODs in Allium Sativum appear to be Cu, Zn-SOD isoenzymes. The isoelectric analysis indicates that three of the four isoermymes are acidic proteins with isoelectric points at pH 3.5, 3.7 and 4. 0, respectively, and the fourth one is a basic pro-tein with isoeletric point at pH 8. 5.     

Simple and rapid catalytic spectrophotometric determination of superoxide anion radical and superoxide dismutase activity in natural medical vegetables using phenol as the substrate for horseradish peroxidase

The coupling reaction of 4-aminoantipyrine (4-AAP) with phenol using the superoxide anion radical ( ) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, produced by irradiating vitamin B₂ (V_B2) was spectrophotometrically determined at 510 nm. Under the optimum experimental conditions, the relationship between A ₅₁₀ and concentration was linear in the range 9.14×10^–6–1.2×10^–4 mol L^–1. The detection limit was determined to be 1.37×10^–6 mol L^–1. A possible reaction mechanism was discussed. The effect of interferences and surfactants on the determination of was also investigated. The proposed method was applied to determine superoxide dismutase activity in garlic, scallion, and onion with satisfactory results.     

天然蛋白质的模拟酶特性研究及应用

本文评述了天然蛋白质作为模拟酶的研究及生物活性物质分析中的应用进展。引用参考文献76篇。     

超氧负离子在生物发光中的作用

活性氧物种和超氧负离子是生物体内的重要物质,本文通过超氧负离子在生物发光反应中的作用,针对几种不同的典型生物发光体系,综述了超氧负离子参与发光反应的相关理论和实验研究进展.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

The Voltammetric Behavior of Superoxide Dismutase/Catalase Mimics

The voltammetric behavior of the superoxide dismutase/catalase mimics [(N,N′‐bis(salicylidene)ethylenediamine)Mn(III)]Cl (or salenMn(III) chloride) 1 and [(N,N′‐bis(3‐methoxysalicylidene)ethylenediamine)Mn(III)]Cl (or 3,3′‐methoxysalenMn(III) chloride) 2 in acetonitrile is described. Both compounds show quasi‐reversible one‐electron reductions to the Mn(II) compound. Electroanalytical techniques are used to follow the reaction between superoxide and 1 and 2 and it is shown that it is the reduced Mn(II) compounds which scavenge superoxide. It is also shown that both compounds electrocatalytically generate superoxide in the presence of dissolved dioxygen. The rate constant for this reaction was determined for both compounds using microelectrode steady state voltammetry. A general reaction scheme for interactions between these compounds and both dissolved dioxygen and superoxide is proposed.     

Electronic and Functional Structure of Copper in Plant Cu/Zn Superoxide Dismutase with Combined Site-directed Mutagenesis and Electron Paramagnetic Resonance

Arabidopsis thaliana copper-zinc superoxide dismutase 1 (AtSOD1) is a typical metalloenzyme conferring cellular protection against the excessive accumulation of toxic reactive oxygen species, and is therefore considered as a critical protein. However, the structure and function of the vital amino acids around the active site of AtSOD1 remain poorly understood. Herein, the coordinated geometry of the catalytic center in AtSOD1 was reconstructed by electron paramagnetic resonance (EPR) technique, and it was found to be composed of copper and four histidine (H) residues using site-directed mutagenesis. Analysis of the mutants showed that H45 and H62 play essential roles in the catalytic reaction, and H119 plays an accessary role in facilitating substrate or proton transfer. The results indicated that the redox change of the Cu ion and the overall enzymatic activity of the protein were sustained by the H45-Cu-H62 core structure. In contrast, the residue H47 showed nearly no effect on the SOD catalytic activity. These data should contribute to a deeper understanding of the catalytic mechanism of the enzyme, and provide a new approach for the effective molecular modification of copper/zinc SODs to facilitate further research in this field.     

低硒大骨节病区粮对大鼠组织中超氧化物歧化酶和脂质过氧化物的影响

应用低硒大骨节病区粮，病区粮加硒和非病区粮饲料喂养大鼠，测定大鼠血清以及心、肝组织中的超氧化物歧化酶(SOD)的活性和肝组织中脂质过氧化物(TBA)含量，观察低硒大骨节病区粮对大鼠体内脂质代谢的影响，结果表明，病区粮组肝组织TBA较病区粮加硒组与非病区粮组显著升高，病区粮加硒组与非病区粮组肝TBA含量无显著性差异，SOD活性变化不明显，这些说明大鼠体内脂质代谢紊乱似与低硒有关，加硒可改善这种代谢。     

Determination of antioxidant capacity of papaya fruit and papaya-based food and drug integrators,using a biosensor device and other analytical methods

Abstract  Antioxidant and radical-scavenging properties are alleged to form the basis of the therapeutic properties attributed to the papaya and papaya-based food and drug integrators that have recently appeared on the market. In the present research a biosensor-based method was used to determine the antioxidant capacity of papaya fruit as is, that of several food and drug integrators, and lastly of pure papain alone. Results are compared using a reference spectrofluorimetric method and two other spectrophotometric methods. In addition pro-oxidant properties of the same samples were also checked using two colorimetric methods. Graphical abstract       

Fabrication of Superoxide Dismutase (SOD) Imprinted Poly(ionic liquid)s via eATRP and its Application in Electrochemical Sensor

Superoxide dismutase (SOD) plays an important role in nearly all living cells. In this study, SOD imprinted poly(ionic liquid)s (SIPILs) were prepared on the surface of the bare Au electrode modified with nano‐palladium and nano‐gold (Au/nPd/nAu/SIPILs). SIPILs was synthesized with 1‐vinyl‐3‐propyl imidazole sulfonate ionic liquids as functional monomers via electrochemically mediated atom transfer radical polymerization (eATRP) catalyzed by SOD. The Au/nPd/nAu/SIPILs was examined by cyclic voltammetry (CV), scanning electron microscope (SEM), energy‐dispersive spectrometer (EDS) and X‐ray photoelectron spectroscopy (XPS). The Au/nPd/nAu/SIPILs was also used as an electrochemical sensor to determine SOD by differential pulse voltammetry (DPV). Under the optimal conditions, the detection range of SOD was from 1.0×10^?8 to 1.0×10² mg L^?1 with a limit of detection of 8.90×10^?9 mg L^?1 (S/N=3). Compared with other methods, the sensor based on SIPILs had the broader linear range and lower detection limit.     

罗丹明6G-I~-作荧光探针研究吲哚乙酸-辣根过氧化酶催化体系中活性氧的产生机理

以罗丹明6G-I-为荧光探针,探讨了辣根过氧化酶催化吲哚乙酸体系中活性氧物种的生成,发现在此体系中可产生单线态氧、超氧、羟基自由基和过氧化氢等活性氧物种.利用活性氧氧化I-成I3-后与罗丹明6G形成离子缔合物将导致其荧光猝灭的现象,可实现活性氧物种总量的测定.相应吲哚乙酸浓度在0.21～262.8μg/mL范围内催化产生的活性氧的总量与荧光猝灭强度△F呈良好的线性关系.反应动力学研究表明,辣根过氧化酶催化吲哚乙酸产生活性氧的反应是一级反应.