谷胱甘肽测定方法研究进展

对分光光度法、酶法、荧光法、电化学法以及和HPLC等测定方法的原理、发展和应用作了详细叙述,同时对其它方法也作了简单介绍,最后对谷胱甘肽测定方法的发展作了展望。     

不同敌百虫抗性淡色库蚊幼虫中谷胱甘肽和谷胱甘肽转移酶的比较

本文比较了三种不同敌百虫抗性淡色库蚊的谷胱甘肽和谷胱甘肽转移酶的活力。结果表明谷胱甘肽含量随着从幼虫、蛹、成虫的发育面逐渐增加,敌百虫抗性品系比敏感晶系有明显高的谷胱甘肽转移酶的活力,同时敌百虫处理幼虫导致谷胱甘肽含量下降。推测敌百虫抗性可能同谷胱甘肽和谷胱甘肽转移酶活力增高有关。     

飞蝗解毒酶系活力测定方法

飞蝗Locusta migratoria是重要的农业害虫,代谢抗性是飞蝗主要的农药抗性机制之一。与代谢抗性相关的解毒酶系主要有:非专一性酯酶系(Non-specficesterases,ESTs)、谷胱甘肽S-转移酶系(Glutathione S-transferases,GSTs)和细胞色素P450单加氧酶系(Cytochrome P450 monooxygenases,P450s),解毒酶系活力的测定是研究飞蝗农药代谢机制的重要途径。本文详细介绍了飞蝗解毒酶系的测定方法,为蝗虫及其他昆虫解毒酶系的测定提供参考。     

植物谷胱甘肽的生物合成及其生物学功能

谷胱甘肽(glutathione,GSH)是硫酸根还原同化途径中主要的含硫非蛋白终端产物,在生物中以还原型谷胱甘肽(reduced glutathione,GSH)和氧化型谷胱甘肽(oxidized glutathione,GSSG)存在。因其在植物体中的广泛存在和独特的还原能力得到广泛关注。本文从谷胱甘肽在植物体内的生物合成,谷胱甘肽的区划、运输和降解以及在非生物胁迫条件下的生物学功能等方面论述了近年来国内外对谷胱甘肽的研究进展。     

谷胱甘肽的研究与应用

谷胱甘肽的研究与应用刘振玉（天津体育学院基础部,天津３００３８１）关键词还原型谷胱甘肽,氧化型谷胱甘肽谷胱甘肽（ＧＳＨ）是由谷氨酸、半胱氨酸和甘氨酸形成的三肽化合物,在生物体内有许多重要作用。合成谷胱甘肽的第一步是在谷氨酸的γ－羧基与半胱氨酸的氨基之...     

溶氧及pH对产朊假丝酵母分批发酵生产谷胱甘肽的影响

在7 L发酵罐中研究了溶氧和pH对产朊假丝酵母分批发酵生产谷胱甘肽的影响。结果表明,当葡萄糖浓度为30 g/L且通气量控制在5 L/min时,搅拌转速达到300 r/min即可满足细胞生长和谷胱甘肽合成对溶解氧的需求。不同pH控制方式对谷胱甘肽分批发酵的影响有较大差异。不控制pH时,细胞干重和谷胱甘肽产量比控制pH为55的发酵分别低27%和95%,且有50%的谷胱甘肽向胞外渗漏。研究了将pH控制在4.0、4.5、5.0、5.5、6.0和6.5的谷胱甘肽分批发酵过程,发现在pH 5.5时谷胱甘肽总产量最高。用前期研究建立的动力学模型模拟了不同pH (4.0～6.5)下的分批发酵过程,并从动力学角度解释了pH对细胞生长和谷胱甘肽合成的影响。     

应用球面对称设计方法优化谷胱甘肽生物合成条件

研究了提高细胞内谷胱甘肽质量分数的方法。谷胱甘肽的总产量与细胞的数量和细胞内谷胱甘肽质量分数有关,通过添加底物和刺激物可以促进谷胱甘肽生物合成,提高胞内谷胱甘肽的质量分数。实验考察了高渗刺激物与前体氨基酸对胞内谷胱甘肽质量分数的影响,并应用球面对称设计优化了实验条件,使得胞内谷胱甘肽质量分数达4.47%,产量达257.3mg.L-1,分别比优化前提高了约69.3%和75.7%。     

变温对鱼类抗氧化防御影响的Meta分析

检索中国期刊全文数据库(1994.1～2009.8)、万方数据库(1980.1～2009.8)、维普数据库(1989.1～2009.8),以及Scopus(1960.1～2009.8)、Elsevier(52009.8)、SpringerLink(52009.8)和Blackwell(52009.8)数据库,系统收集涉及温度变化导致鱼类组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)、谷胱甘肽还原酶(GR)、还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)和丙二醛(MDA)变化的相关研究,对纳入23篇文献进行了数据提取和Meta分析,并系统评价变温对鱼类抗氧化防御的影响。除GSSG由于研究数太少不能分析外,Meta分析显示:升温显著提高SOD(标准化均数差SMD=1.0,95% CI=0.4～1.7,P=0.001)和GPX(SMD=0.4,95% CI=0.1～0.7,P=0.005)活力,降温显著下调GPX(SMD=-0.9,95% CI=-1.7～-0.1,P=0.025)和GR(SMD=-1.6,95% CI=-2.5～-0.8,P<0.001)活力。升降温对CAT活力和GSH均无显著影响(P>0.05),但都会显著增加MDA水平(SMD=1.2～1.4,P<0.006)。不同鱼类、组织和测定方法不是引起研究异质性的主要因素,但试验设计的变温幅度是产生SOD、CAT和MDA研究间异质性的主要因子,实验开始温度也会引起GSH研究间的异质性。     

亚硒酸钠对小麦幼苗中谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及谷胱甘肽含量的影响

用1.0 mg·L-1的亚硒酸钠根施小麦幼苗,测定亚硒酸钠对谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及还原性谷胱甘肽含量的结果表明,外源亚硒酸钠对麦苗地上部的谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性均有诱导作用,使麦苗体内的谷胱甘肽含量水平增加.     

植物谷胱甘肽转移酶的结构与功能及其基因表达

植物的谷胱甘肽转移酶是一个多基因家族。文章介绍了谷胱甘肽转移酶的类型、结构、功能和基因表达的研究进展。     

Purification of glutathione reductase from gerbil liver in two steps

A new method for the isolation of glutathione reductase which successively utilizes chromatography on 2'-5'-ADP-Sepharose 4B and DEAE-Sepharose CL 6B, is described. With these two steps, it was possible to purify to homogeneity the glutathione reductase from gerbil liver. Some molecular properties of the purified enzyme are reported.     

薄层色谱和高效液相色谱联用测定暗纹东方鲀肌肉中肌肽和谷胱甘肽的含量

通过薄层色谱(TLC)与高效液相色谱(HPLC)联用技术鉴定了暗纹东方鲀(Takifugu obscurus)肌肉中存在肌肽和谷胱甘肽,同时利用高效液相色谱法测定了肌肽和谷胱甘肽(GSH)的含量。薄层层析采用的展开剂为正丁醇:乙酸:水(4∶2∶1),层析板为硅胶板。高效液相色谱利用Kromasil C18反相柱分析,流动相为10%的纯乙腈和90%含有0.05%三氟乙酸的超纯水。结果表明:通过薄层色谱和高效液相色谱鉴定了暗纹东方鲀肌肉中肌肽和谷胱甘肽,其中暗纹东方鲀肌肉中肌肽含量约213μg/g(鲜重),还原性谷胱甘肽含量约211μg/g(鲜重)。本法样品无需衍生,操作简便,适合于暗纹东方鲀肌肉中肌肽和谷胱甘肽的测定。本文为生物体内肌肽和谷胱甘肽的研究提供借鉴意义。     

Determination of content of metallothionein and low molecular mass stress peptides in transgenic tobacco plants

Phytoremediation is a process that utilizes plants to remove, transfer, stabilize, or destroy pollutants in soil, sediment, and groundwater. Plants used for such purposes have several requirements. Genetic engineering these plants could be an effective tool used to acquire features needed for such purposes within a substantial amount of time. This paper aims to utilize electrochemical techniques to analyze transgenic tobacco and, thus, to reveal their heavy metals phytoremediation potential. Total thiol and metallothionein (MT) quantities were determined in the control and transgenic tobacco plants. The total content of thiols in transgenic plants varied within the range of 561 to 1,671 μg g⁻¹. Furthermore, the determination of MT was done on transgenic tobacco plants. The level of human MT in transgenic tobacco plants varied between 25 and 95 μg g⁻¹. However, a plant cell protects itself by synthesizing low molecular mass thiols such as reduced glutathione and phytochelatins to protect itself against heavy metals toxicity. The most important thiols, cysteine (Cys), glutathione (GSH), oxidised glutathione (GSSG) and phytochelatin 2 (PC2), were determined in the non-transgenic and transgenic tobacco plants by high performance liquid chromatography with electrochemical detection. Tobacco plants synthesizing the highest amount of metallothionein have the highest basal level of phytochelatin 2 as well as reduced glutathione and free cysteine. It clearly follows from the results obtained that the biosynthesis of particular thiols is mutually linked, which contributes to a better protection of a transgenic plant against heavy metals effects.     

Reversed-phase chromatographic method for specific determination of glutathione in cultured malignant cells

A chromatographic method for the specific determination of glutathione in malignant cell lines is described. The method is based on the ability of glutathione-S-transferase to specifically and quantitatively conjugate glutathione to 1-chloro-2,4-dinitrobenzene and chromatographic quantitation of the resultant conjugate, dinitrophenyl-S-glutathione, by reversed-phase liquid chromatography. The assay can be performed on 20 000 g supernatants of cell homogenates without acid extraction. 2-Mercaptoethanol, a sulfhydryl compound often used as a thiol-protective agent to preserve enzymatic activities of a number of enzymes, did not interfere with glutathione determination by this method. The dinitrophenyl-S-glutathione isolated from either standard glutathione samples or from cell homogenates was shown to be identical to authentic dinitrophenyl-S-glutathione using mass spectrometry. Recovery of glutathione in standard samples by the current method was identical to that determined using 5,5′-dithiobis(2-nitrobenzoic acid). Exogenous glutathione added to supernatants of cell homogenate in the presence or absence of 2-mercaptoethanol was also completely recovered.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Analysis of saliva for glutathione and metabolically related thiols by liquid chromatography with ultraviolet detection

Summary. A method for simultaneous determination of glutathione and its precursors cysteine, cysteinylglycine and homocysteine in saliva is presented. The procedure involves reductive conversion of disulfides to thiols, derivatization to their 2-S-quinolinium derivatives with 2-chloro-1-methylquinolinium tetrafluoroborate and separation and quantitation by reversed-phase ion-pairing high performance liquid chromatography with ultraviolet detection at 355 nm. The calibration performed with saliva samples spiked with thiol disulfides, within the practical concentration ranges, showed linear response of the detector. The method applied to the saliva samples donated by volunteers showed mean concentration (SD, n = 8) of cysteine, cysteinylglycine, glutathione and homocysteine: 26.5 (31.6), 6.05 (5.12), 16.97 (7.68), 3.64 (1.34) nmol/ml respectively.     

Determination of eleven small selenium species in human urine by chromatographic-coupled ICP-MS methods

BackgroundThe determination of various selenium species in urine enables a specific biomonitoring of the exposure to different selenium compounds.MethodsFor this task a coupling of three chromatographic techniques with ICP-MS was developed for the separate quantification of eleven species in urine. The first procedure was based on reverse phase chromatography and was designed for the separate determination of methyl-2-acetamido-2-deoxy-1-seleno-b-d-galactopyranoside (SeSug1), methyl-2-acetamido-2-deoxy-1-seleno-b-d-glucopyranoside (SeSug2), selenomethionine (SeMet), methylselenocysteine (MeSeC), seleno-D,L-ethionine (SeEt), methylselenic acid (MeSeA) and methylselenoglutathione (MeSeG); the second procedure was based on anion exchange chromatography and measured selenate (Se (VI)) and selenite (Se (IV)); the third procedure was based on cationic exchange chromatography and determined methyl-2-amino-2-deoxy-1-seleno-b-d-galactopyranoside (SeSug3) and the trimethylselenium ion (TMSe). A fourth method for the more sensitive determination of TMSe was upgraded by an on-line after-column reaction process.ResultsThe validation of the methods yielded sensitive detection limits of the species between 0.03 and 0.10 μg Se/L. For TMSe a detection limit of 0.02 μg Se/L resulted by the fourth method. An intra-day precision of 2.7–10.6% and a relative recovery between 87 % and 108 % confirm the robustness of the methods.ConclusionThe developed procedures enable a separate and sensitive determination of eleven selenium species in urine and thus permit the exploring of metabolic factors in the general population and particularly exposed individuals.     

昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ,ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经１００００ｇ和１０００００ｇ分级离心;取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４）,四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４）,铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质,通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明,采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰,在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带,分子量为２４ｋＤ,２４．５ｋＤ左右;Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰,呈现出一条带,分子量为２４ｋＤ左右;ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰,呈现出一条带,分子量为２３ｋＤ左右     

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议