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.     

具有谷胱甘肽过氧化物酶活性的催化抗体的研究Ⅰ．谷胱甘肽特征全抗原的合成、表征及性质

用２，４－二硝基氯苯保护谷胱甘肽的巯基制出半抗原，再通过戊二醛共价反应使其与牛血清白蛋白表面的氨基结合，经超胶ＡｃＡ５４凝胶层析纯化，制备出有较强免疫原性的谷胱甘肽全抗原。用元素分析、红外光谱及核磁共振表征了半抗原的结构。电泳分析得全抗原分子量平均为８７０００道尔顿。光谱分析及圆二色谱研究表明其有较强的可见、紫外及荧光特征吸收，且抗原结构的紧密性增强。     

Glutathione Isopropyl Ester Reduces UVB-lnduced Skin Damage in Hairless Mice

Abstract— The protective effect of administration of glutathione (GSH) isopropyl ester on photodamage, such as lipid peroxidation, inflammation and tumorigenesis induced by UV exposure (290–400 nm, max. 312 nm), was investigated using hairless mice. Pretreatment with 20 mg/kg GSH isopropyl ester prevented the increases of thiobarbituric acid-reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, which were caused by a single dose (15 kj/m²) of UV irradiation. The level of epidermal GSH in skins of the GSH ester-treated mice was maintained within normal limits. When mice were exposed to UV at a dose of 2 kj/m², three times weekly, skin tumors developed in all of them after 25 weeks. The formation of skin tumors was significantly inhibited by administration of 10 mg/kg GSH ester prior to each UV irradiation for 25 weeks. Moreover, the increases of cutaneous TBARS and serum sialic acid in the tumor-bearing mice were also prevented by continuous pretreatment with GSH ester. Even after 24 weeks, the epidermal GSH content of the pretreated mice was mostly retained compared to nonirradiated mice. However, administration of GSH prior to acute or chronic UV irradiation had no effect on the UV-induced damage. The present results suggest that the protection from photo-damage afforded by pretreatment with GSH ester is due to maintenance of a normal GSH level.     

Design and efficient synthesis of novel haptens and complete antigens for the AOZ, a toxic metabolite of furazolidone

A good strategy was brought forward for designing efficient haptens and complete antigens for 3-amino-2-oxazolidinone (AOZ). Haptens designed newly were achieved facilely in good yield by using LiCI-N(Et)_3 as new catalysis system,the structure of which was elucidated by spectroscopy analysis,such as NMR and MS.Novel antigens for AOZ were prepared successfully by convenient active ester method.The ratios of haptens 3 and 4 to carrier proteins were proven respectively as 41:1(5a),39:1(6a),11:1(5b) and 9:1(6b) by trinitrobenzene sulfonic acid(TNBS) method.The results of indirect competitive ELISA (ic-ELISA) of antiserums indicated that the haptens with a short unsaturated side chain can evoke specific immune response effectively.     

Study of Oxidation of Glutathione Treated with Hypochlorous Acid by Capillary Electrophoresis

Glutathione (GSH) protects human and animal's cells by the exchange with reversible oxidized glutathione (GSSG) when cells meet with oxidants, such as hydrogen peroxide (H2O2) and lipid peroxide. But when GSH meets with hypochlorous acid (HOCl), via the myeloperoxidase-catalysed oxidation of chloride by H2O2, the amount of GSSG formed does not account for all the GSH lost, which is quite different from that we has investigated in the reaction of GSH with H2O21. Prutz proposed the fo…     

Substrate Profiling of Glutathione S‐transferase with Engineered Enzymes and Matched Glutathione Analogues

The identification of specific substrates of glutathione S‐transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G‐W40A‐R41A mutant (GST‐KWR) of the mu‐class glutathione S‐transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH‐R1) as the cosubstrate. The GSH‐R1 conjugation products can react with an azido‐based biotin probe for ready enrichment and MS identification. Proof‐of‐principle studies were carried to detect the products of GSH‐R1 conjugation to 1‐chloro‐2,4‐dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.     

Amperometric determination of glutathione and cysteine on a Pd-IrO(2) modified electrode with high performance liquid chromatography in rat brain microdialysate

A Pd/IrO(2) co-electrodeposited glassy carbon electrode was prepared and the electrochemical behavior of glutathione (GSH) at this chemically modified electrode (CME) has been studied by cyclic voltammetry (CV). The results indicated that the modified electrode efficiently exhibited electrocatalytic oxidation for GSH with relatively high sensitivity, stability, and long-life. Coupled with high-performance liquid chromatography (HPLC), the Pd/IrO(2) modified electrode was utilized for the electrochemical detection (ECD) of the thiocompounds, glutathione and cysteine (Cys). The peak currents were linear with the substance concentrations in the range of 1.0 x 10(-5) mol L(-1) to 8.0 x 10(-4) mol L(-1) for GSH and 4.0 x 10(-6) mol L(-1) to 2.0 x 10(-4) mol L(-1) for Cys. The detection limits were 2.0 x 10(-6) mol L(-1) for GSH and 5.0 x 10(-7) mol L(-1) for Cys with S/N of 3. The method has been successfully applied to assess the contents of GSH and Cys in rat brain microdialysates.     

High-performance liquid chromatography/chemiluminescence determination of biological thiols with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide

The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH group. The labelling reaction was carried out at 60 degrees C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODS-80 column (150 x 4.6 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23 +/- 0.15 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.     

Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfide

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.     

用磷酸二异辛酯/正辛醇反胶束从面包酵母粗提液中萃取谷胱甘肽

采用磷酸二异辛酯（P204）/正辛醇反胶束液从面包酵母粗提液中萃取还原型谷胱甘肽(GSH),研究了各种因素对萃取率的影响以及水相pH值和P204浓度与GSH萃取率关系的数学模型。正交实验确定,GSH的最佳萃取条件是水相pH值2.0,K+浓度0.1 mol•L-1,萃取时间15 min,P204浓度0.16 mol•L-1,GSH单次的萃取率为33.1%,2次萃取率达到49.7%。     

Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to-noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product that could possibly affect the quantification of GSH and GSSG.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

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.     

电化学间接测量法研究谷胱甘肽在多壁碳纳米管和活性炭上的吸附

选取谷胱甘肽(GSH)作为小分子代表物,利用Cr(VI)与GSH的相互作用,即K2Cr2O7/H2SO4溶液加入GSH前后Cr(VI)还原峰电流值的差异,通过差分脉冲伏安法(DPV)的测定,可以间接测出GSH的含量.尝试将这种电化学间接测量法应用于研究多壁碳纳米管(MWCNTs)与商业活性炭(AC)对GSH的吸附行为.利用该方法可以确立GSH在二者的吸附量(Q)与吸附平衡浓度(Ce)的关系,绘制吸附等温曲线.根据Langmuir方程和Freundlich方程的拟合分析,证明了与商业活性炭相比,GSH在MWCNTs上的吸附更倾向Freundlich模型,即多分子层吸附.利用扫描电子显微镜(SEM)和透射电子显微镜(TEM)进行形貌表征,发现MWCNTs具有发达的堆积孔结构,有利于GSH小分子内扩散和吸附,与实验结果一致.     

Profiling the Concentration of Reduced and Oxidized Glutathione in Rat Brain Using HPLC/DAD Chromatographic System

This study aimed to develop a HPLC/DAD method in order to determine and quantify the reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in rat brain. Due to the presence of the thiol group (-SH), GSH can interact with the Ellman′s reagent (DTNB), with which it forms a reaction product through which the level of GSH can be quantified, using the DAD detection system. Chromatographic separation was achieved after a derivatization process by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 2.5) (B). The compounds of interest were detected at 330 nm using a chromatographic C8 column. The method of determination met the validation criteria, specified by the regulatory bodies. The applicability of the method was demonstrated in a chronic toxicology study of central nervous system (CNS), following different treatment regimens with haloperidol.     

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.     

Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR

The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real‐time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U‐¹³C]‐labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS‐producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti‐glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up‐regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox‐dependent signaling pathways and drug resistance involving ROS.     

SRXRF study of trace elements in hippocampus of pup rats after prenatal and postnatal exposure to low-level mercury

The damage in the pup rat brain with low-level mercury exposure, and the concentration variation of trace elements in the rat hippocampus was determined by synchrotron radiation X-ray fluorescence technique (SRXRF). Meanwhile, the levels and activities of glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in the hippocampus were also measured. The results showed that the low dose of inorganic mercury prenatal and postnatal exposure could lead to the significant increase of both copper and zinc contents and remarkable decrease of iron content in pup rat brain. Compared to the control group, the activities of antioxidant enzymes such as GSH-Px, SOD, the contents of GSH and MDA in the pup rat hippocampus of mercury-exposed group fell down obviously.     

Imprinted Human Serum Albumin with Antioxidant Activity

In order to create a new mimic of glutathione peroxidase(GPx), bioimprinting was used to generate gluta-thione(GSH) binding site and chemical modification was used to incorporate catalytic group selenocystine(Sec). Human serum albumin(HSA) and S-substituted dinitrophenyl glutathione(GSH-S-DNP) were chosen as the imprinted matrix and imprinting template, respectively, to generate a GSH-imprinted protein(GSH-HSA) by bioimprinting. Sec was incorporated into the GSH-HSA by chemical modification to give a new GP...     

A Multi‐signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine,Homocysteine, and Glutathione

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn‐on for distinguishing Cys, Hcy, and GSH with 108‐, 128‐, 30‐fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.