斜纹夜蛾3种SlitPBPs的组织表达模式分析

性信息素结合蛋白(pheromone binding proteins,PBPs)能够与性信息素分子结合,从而启动昆虫的寻偶及交配行为。本研究采用半定量RT-PCR技术分别对斜纹夜蛾3种性信息素结合蛋白SlitPBP1、SlitPBP2和SlitPBP3基因在雌、雄虫不同组织的基因表达模式进行了比较分析。组织表达模式结果表明,SlitPBP1基因只在雌、雄虫触角和雌虫前足中表达而在其他组织中无表达;SlitPBP2基因在雌虫的触角、喙、前足、中足、后足、翅膀和雄虫的触角、喙、前足、翅膀中均有表达,且在同一部位,SlitPBP2基因在雌虫的表达量均高于雄虫相应部位的表达量;除雌、雄虫的胸部外,SlitPBP3在雌、雄虫的触角、喙、前足、中足、后足、腹部和翅膀中均有较高水平的表达。可见,SlitPBP1、SlitPBP2和SlitPBP3基因在斜纹夜蛾不同组织的表达模式各不相同。本研究结果可为深入研究不同SlitPBPs蛋白在斜纹夜蛾生长发育过程中的不同生理功能提供科学参考。     

桃蛀螟成虫Orco嗅觉受体基因的克隆及组织表达谱分析

【目的】克隆桃蛀螟Conogethes punctiferalis (Guenée)的Orco嗅觉受体基因, 并研究其在不同组织的表达谱。【方法】利用PCR技术克隆桃蛀螟触角Orco基因, 对该基因编码的氨基酸序列进行生物信息学分析, 并利用荧光定量PCR技术分析该基因在的表达量。【结果】获得桃蛀螟成虫Orco的cDNA全长序列, 并命名为CpunOrco（GenBank登录号： JX101681）。该基因的开放阅读框全长1 425 bp, 编码475个氨基酸, 序列中有7个跨膜区。对桃蛀螟成虫不同组织中CpunOrco的荧光定量PCR结果表明, CpunOrco主要在触角和下颚须中表达, 雄虫触角中的表达量高于雌虫, 并且该基因在其他组织中也有一定的表达。【结论】本研究明确了该嗅觉受体基因在桃蛀螟成虫不同组织内的表达水平, 为进一步研究其功能提供了理论依据。     

绿盲蝽气味受体基因AlucOR40的克隆及功能研究

【目的】从绿盲蝽Apolygus lucorum触角中克隆气味受体基因Aluc OR40,研究该气味受体基因在绿盲蝽不同组织中的表达水平,然后对该基因的功能进行研究,为理解绿盲蝽的嗅觉识别机制提供理论基础。【方法】在绿盲蝽成虫触角转录组测序与分析的基础上,通过PCR技术克隆得到气味受体基因Aluc OR40的全长序列。用半定量RT-PCR研究该基因在雌雄虫不同组织中的表达水平。然后通过爪蟾卵母细胞体外表达该基因,并结合双电极电压钳检测了该气味受体对60种气味分子包括绿盲蝽性信息素组分和植物挥发物的反应。然后,利用触角电位技术测定羽化后3 d的绿盲蝽成虫对Aluc OR40的气味配体的触角电位反应。【结果】克隆了Aluc OR40(Gen Bank登录号:KU886190)。Aluc OR40编码398个氨基酸,蛋白具有7个跨膜结构域,N末端位于胞内,C端位于细胞外,符合昆虫气味受体的典型特征。半定量RT-PCR的结果显示,Aluc OR40在触角中特异表达,且在雄虫触角中的表达水平高于雌虫。双电极电压钳记录结果显示,Aluc OR40只对反-2-己烯醇一种气味分子具有特异反应。EAG实验结果表明,羽化后3 d的绿盲蝽雌雄虫都对反-2-己烯醇有反应,且雄虫触角的EAG反应显著高于雌虫。【结论】结果提示Aluc OR40参与了绿盲蝽对反-2-己烯醇的识别过程,推测其在绿盲蝽交配过程中雄虫对雌虫的识别中发挥作用。     

烟夜蛾性信息素结合蛋白2(PBP2)基因的克隆、序列分析与时空表达

性信息素结合蛋白（pheromone binding proteins, PBPs）在昆虫雌雄间信息交流中起着重要作用。 本研究利用RT-PCR和RACE方法, 从烟夜蛾Helicoverpa assulta (Guenée)雄虫触角中克隆了性信息素结合蛋白2基因的开放阅读框及3′末端序列, 该基因被命名为HassPBP2(GenBank登录号为EU316186)。克隆和测序结果表明, HassPBP2开放阅读框全长450 bp, 编码149个氨基酸残基, 推测编码蛋白的分子量为16.9 kD, 等电点为5.56。HassPBP2基因结构分析表明, 该基因由3个外显子和2个内含子组成, 内含子的长度分别为90和261 bp。氨基酸序列联配分析表明, 此序列具有气味结合蛋白的典型特征, 与其他鳞翅目昆虫PBPs的一致性在34%~91%之间, 其中与棉铃虫Helicoverpa armigera PBP2和烟芽夜蛾Heliothis virescens PBP2的序列一致性高达91%。时间表达和组织表达分析显示, HassPBP2在卵期、幼虫期和蛹早期不表达, 在蛹中期开始表达, 并一直持续到成虫中期, 且只在雌、雄成虫触角中表达。     

瓜实蝇嗅觉受体基因的克隆及表达谱分析

昆虫的嗅觉受体是一个高度变异的蛋白家族, 其中一类Or83b嗅觉受体在不同昆虫体内高度保守, 在昆虫的行为调控过程中起到十分重要的作用。为进一步探讨Or83b受体的功能, 本研究利用RT-PCR和RACE方法克隆获得瓜实蝇Bactrocera cucurbitae (Coquillett) Or83b-like受体的全长cDNA序列, 命名为BcucOr83b-like（GenBank登录号： HM745934）。测序结果表明, BcucOr83b-like开放阅读框全长1 422 bp,编码473个氨基酸残基。氨基酸序列比对表明, 此序列具有Or83b受体的典型特征, 序列中具有7个跨膜区和高度保守的C端区域。BcucOr83b-like与其他昆虫的Or83b具有较高的氨基酸序列一致性, 其中与桔小实蝇Bactrocera dorsalis（Hendel）Or83b的序列一致性高达99.6%。对该基因在瓜实蝇成虫不同组织和发育时期表达量的荧光定量PCR分析表明, BcucOr83b-like主要在瓜实蝇成虫触角中表达, 头部（去除触角）、 雌虫前足和翅中也有较高的表达; 瓜实蝇在各个发育时期的表达水平不同, 在刚羽化雌成虫中的表达量最高。本研究为深入研究瓜实蝇Or83b受体的功能提供了理论依据。     

棉铃虫感觉神经元膜蛋白基因克隆和表达

从棉铃虫Helicoverpa armigera触角中克隆了一条全长1 690 bp的cDNA序列,该序列阅读框全长1 572 bp,编码523个氨基酸残基,序列中有2个跨膜区,具有昆虫感觉神经元膜蛋白（sensory neuron membrane protein, SNMP）的典型特征。SNMP与已报道的其他昆虫的感觉神经元蛋白的氨基酸序列有很高的同源性。半定量RT-PCR研究结果显示,SNMP在棉铃虫中不仅在触角中表达,也在去掉触角的头、足中表达。但是在触角中的表达量最高,在雌雄触角中的表达量差异不显著。在喙、下颚须和下唇须中也有表达。SNMP在卵、蛹和成虫体内也都有表达,但在卵中表达量相对较低。将SNMP编码区克隆到表达载体pET21b中,成功地进行了原核表达,表达出带有6个组氨酸标签的重组蛋白。     

二化螟Minus-C气味结合蛋白的分子克隆及功能鉴定

气味结合蛋白（odorant binding proteins, OBPs）在昆虫对寄主气味的感受中起重要作用, 但有关Minus-C OBP及其功能的报道很少。本研究通过基因组数据分析并利用RACE技术, 克隆和鉴定了二化螟Chilo suppressalis (Walker)的一个Minus-C OBP基因, 命名为CsupOBP1（GenBank登录号： KC492498）。CsupOBP1基因的开放阅读框长423 bp, 编码141个氨基酸, 其中N端的18个氨基酸为预测的信号肽序列, 成熟蛋白序列中具有4个保守的半胱氨酸位点。实时定量PCR分析显示, 该基因在幼虫头部及成虫雌雄足、 翅和雄性触角等化感组织中高表达, 其中在雄虫触角内的表达量显著高于雌虫触角。利用荧光竞争结合实验对CsupOBP1重组蛋白与38种化合物的结合特性的测定表明, 重组CsupOBP1与β 紫罗兰酮的结合能力最强（Ki=9.53 μmol/L）。触角电位测定表明, 二化螟成虫可对β-紫罗兰酮产生显著的触角电生理反应, 但雄虫反应明显强于雌虫, 与结合试验及雄虫触角中CsupOBP1的表达量显著高于雌虫触角的测定结果相一致。鉴于β-紫罗兰酮是水稻等植物中普遍存在的一种芳香气味组分, 推测CsupOBP1可能通过对该气味的结合和运输, 从而在二化螟对寄主植物的嗅觉定向中起作用。     

棉铃虫触角羧酸酯酶HaCXE6基因的克隆、序列分析与mRNA表达

为研究羧酸酯酶(carboxylesterase,COE)在棉铃虫Helicoverpa armigera(Hübner)触角中的生理功能,采用RT-PCR、RACE方法从棉铃虫雄蛾触角中克隆得到一条COE基因全长序列(GenBank No.JX306730),命名为HaCXE6。HaCXE6基因读码框1 623 bp,编码540个氨基酸,预测蛋白质分子量61.02 ku,等电点8.06。氨基酸序列比对分析表明,HaCXE6与海灰翅夜蛾Spodoptera littoralis触角羧酸酯酶CXE6一致性最高,为68%;棉铃虫不同功能羧酸酯酶聚类分析显示HaCXE6与气味降解酶CCE006a最相近。与其它昆虫触角羧酸酯酶多序列比对分析发现,HaCXE6具有酯酶活性必须的催化残基Ser199,Glu323,His434,也保持着α/β-酯酶家族的特征基序Gly-xSer-x-Gly。不同组织和发育时期该基因荧光定量PCR分析结果表明,HaCXE6在雌、雄蛾各个部位均有表达,雌蛾腹部与雄蛾足中表达量最高;卵至成虫各个时期均有表达,蛹中表达量最高。     

棉铃虫离子型受体基因HarmIR8a的克隆及表达定位

【目的】昆虫离子型受体(ionotropic receptors, IRs)是新发现的一类化学感觉受体,对昆虫感受环境中酸类和胺类等物质发挥着重要作用。基因克隆、序列比对以及表达定位的研究将有助于初步解析棉铃虫Helicoverpa armigera离子型受体的功能。【方法】本研究在棉铃虫触角转录组测序和分析的基础上,利用PCR技术克隆了一个离子型受体基因的全长序列,并进行序列结构预测等分析;利用荧光定量PCR技术对该基因在棉铃虫雌、雄成虫不同组织中的表达量进行了分析;同时利用原位杂交技术检测了该基因在棉铃虫雄虫触角中的表达定位。【结果】克隆获得棉铃虫HarmIR8a基因全长cDNA序列(GenBank登录号:MH638313),开放阅读框为2 688 bp,编码895个氨基酸,预测有3个跨膜区域。HarmIR8a氨基酸序列包含了一个氨基末端区域(amino terminal domain, ATD),一个由S1和S2两部分构成的配体结合域(ligand binding domain, LBD),一个孔环(pore loop, P),3个跨膜区(M1, M2和M3)和一个胞内C末端(C terminus)。荧光定量PCR结果显示,HarmIR8a在棉铃虫雌、雄成虫头(除去附器)和触角等组织中均有表达,而且在触角中高表达。棉铃虫雄虫触角原位杂交结果表明,HarmIR8a主要在触角腔锥形感器下表达。【结论】本研究初步探析了HarmIR8a基因的转录水平和表达部位,为进一步研究棉铃虫离子型受体基因的功能和生理作用奠定了基础。     

烟夜蛾性肽受体基因的克隆与表达模式分析

【目的】克隆烟夜蛾Helicoverpa assulta (Guenée)性肽受体基因并分析其表达模式, 为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法, 从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因cDNA全序列。利用荧光定量PCR方法, 分析该基因的表达模式。【结果】序列分析结果显示, 烟夜蛾性肽受体基因cDNA全长2 048 bp, 命名为HassSPR（GenBank登录号: AFH53182.1）。该基因的开放阅读框长1 275 bp, 编码424个氨基酸残基, 序列中含有7个跨膜域结构, 预测分子量和等电点分别为48.6 kDa和9.25。序列比对分析表明, HassSPR与近缘种棉铃虫H. armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%, 与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明, HassSPR在测定的1日龄雌蛾不同组织中均有表达, 以在脑中的表达量最高。时序表达分析表明, 在羽化前1 天至羽化后6日龄雌蛾的信息素腺体中均有表达, 以3日龄表达量最高。雌蛾交配后, HassSPR在性信息素腺体和脑中的表达量显著上调, 而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因HassSPR, 其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。     

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) glutathione S-transferase

A cDNA of glutathione S-transferase (GST) was isolated from a cDNA library of salivary glands of Boophilus microplus. The recombinant protein was purified by glutathione affinity chromatography and assayed upon the chromogenic substrate CDNB. The 864 bp cloned fragment was sequenced and showed an open reading frame coding for a protein of 220 amino acids. Expression of the GST gene was tested by RT-PCR in tick tissues and larvae mRNA. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian class mu GSTs.     

Transcobalamin II expression is regulated by transcription factor(s) binding to a hexameric sequence (TGGTCC) in the promoter region of the gene

An isoenzyme of glutathione S-transferase (adGST) was purified from liver intestine of the seashell (Asaphis dichotoma) by GST-Sepharose 4B affinity chromatography followed by reverse-phase HPLC. The enzyme has a pI value of 4.6 and is composed of two subunits each with a molecular weight of 23kDa. It exhibits different catalytic activities toward the substrates 1-chloro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, ethacrynic acid, and p-nitrophenyl acetate and, fascinatingly, shows high activity toward CDNB. The amino acid composition of adGST was determined and found to be very similar to the Sloane squid GSTs. N-terminal analysis of the first 15 residues of adGST revealed that it has 73% sequence identity with the pig roundworm GSTs. The adGST shows characteristics similar to those of class sigma GSTs, as was indicated by its substrate specificity, N-terminal amino acid sequence, and amino acid composition.     

烟夜蛾精氨酸激酶基因的克隆及mRNA表达分析

为了深入了解精氨酸激酶基因的作用和寻求害虫防治新的分子靶标, 本研究采用RT-PCR和RACE技术, 从烟夜蛾Helicoverpa assulta脂肪体中克隆了精氨酸激酶cDNA序列, 命名为HassAK（GenBank登录号: HQ336337）, 并采用荧光定量PCR测定了HassAK基因在不同发育阶段（4龄幼虫第1天到化蛹第1天）、 不同组织（头部、 中肠、 脂肪体、 体壁和腹足）和不同温度条件下的表达情况。测序和序列分析结果表明, HassAK基因阅读框架全长1 068 bp, 编码355个氨基酸残基, 预测蛋白质分子量和等电点分别为40.0 kD和5.76。氨基酸序列分析表明, 该序列具有精氨酸激酶典型的酶活性部位、 酶活性中心位点和能形成离子偶结构的保守区。序列比对结果表明, HassAK与其他昆虫AK的氨基酸序列具有70%以上的一致性。荧光定量分析结果显示, HassAK基因在幼虫头部、 中肠、 脂肪体、 体壁和腹足均可表达, 其中以腹足和中肠内的表达水平较高。时序表达分析表明, 预蛹期HassAK基因的表达量达到高峰。此外, 高温和低温均诱导HassAK基因的表达, 说明该基因可能参与昆虫抵御外界不良环境。     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Identification and cloning of a key insecticide-metabolizing glutathione S-transferase (MdGST-6A) from a hyper insecticide-resistant strain of the housefly Musca domestica

Strains of the housefly, Musca domestica, highly resistant to organophosphate (OP) and other insecticides are known because they overproduce glutathione S-transferases (GSTs). Previous work has shown that overproduction in these strains involved numerous isozymes with glutathione conjugating activities (Pesticide Biochem. Physiol., 25 (1986) 169; Mol. General Genetics, 227 (1991) 355; J. Biol. Chem., 267 (1992) 1840; Mol. General Genetics, 245 (1994) 236; J. Mol. Evol., 43 (1996) 236). The current work describes the purification and identification of a M. domestica GST isozyme (pI 7.1) broadly specific for substrates from a housefly strain, Cornell-HR, that is highly resistant against OP-insecticides, and the isolation of two new MdGST genes using the antibody made against it. This isozyme, which was identified from amongst more than 20 isoelectric forms of GSTs of the same subunit size, was highly active for conjugating GSH to the model substrate 3,4-dichloronitrobenzne (DCNB). When expressed in Escherichia coli, one of the cloned GSTs, MdGST-6A, produces an enzyme that conjugates glutathione to the insecticides methyl parathion and lindane. On indication that it was the most active isozyme toward several xenobiotics among several MdGSTs tested, we advance the notion that MdGST-6A probably plays an important role in M. domestica Cornell-HR's resistance towards OP-insecticides. MdGST-6A and a second closely related one found in this work, MdGST-6B, are members of the traditional insect class I family (theta-class) and share the greatest homologies with a cluster of Drosophila GSTs on locus 55. In addition to having the unusually broad substrate specificity, the sequence of the new group of enzymes reveals that it has a highly diverged hydrophobic motif in its active site as compared to other class I GSTs from insects.     

Identification and cloning of a key insecticide-metabolizing glutathione S-transferase (MdGST-6A) from a hyper insecticide-resistant strain of the housefly Musca domestica

Strains of the housefly, Musca domestica, highly resistant to organophosphate (OP) and other insecticides are known because they overproduce glutathione S-transferases (GSTs). Previous work has shown that overproduction in these strains involved numerous isozymes with glutathione conjugating activities (Pesticide Biochem. Physiol., 25 (1986) 169; Mol. General Genetics, 227 (1991) 355; J. Biol. Chem., 267 (1992) 1840; Mol. General Genetics, 245 (1994) 236; J. Mol. Evol., 43 (1996) 236). The current work describes the purification and identification of a M. domestica GST isozyme (pI 7.1) broadly specific for substrates from a housefly strain, Cornell-HR, that is highly resistant against OP-insecticides, and the isolation of two new MdGST genes using the antibody made against it. This isozyme, which was identified from amongst more than 20 isoelectric forms of GSTs of the same subunit size, was highly active for conjugating GSH to the model substrate 3,4-dichloronitrobenzne (DCNB). When expressed in Escherichia coli, one of the cloned GSTs, MdGST-6A, produces an enzyme that conjugates glutathione to the insecticides methyl parathion and lindane. On indication that it was the most active isozyme toward several xenobiotics among several MdGSTs tested, we advance the notion that MdGST-6A probably plays an important role in M. domestica Cornell-HR's resistance towards OP-insecticides. MdGST-6A and a second closely related one found in this work, MdGST-6B, are members of the traditional insect class I family (theta-class) and share the greatest homologies with a cluster of Drosophila GSTs on locus 55. In addition to having the unusually broad substrate specificity, the sequence of the new group of enzymes reveals that it has a highly diverged hydrophobic motif in its active site as compared to other class I GSTs from insects.     

Characterization of glutathione S-transferases from day-old chick livers

Glutathione S-transferases (GSTs, EC 2.5.1.18) were isolated from the liver cytosolic fraction of 1 day old Leghorn chicks by S-hexylglutathione and glutathione affinity columns arranged in tandem. After sample loading, the affinity columns were detached from each other and developed separately. Four groups of GSTs (CL 1, 2, 3, and 4) were eluted from the hexylglutathione column, and an additional group of GSTs (CL 2 and 5) was eluted from the glutathione affinity column. CL 2, CL 3, and CL 5 were further purified to homogeneity by chromatofocusing, and the substrate specificities of each group were determined. Fractions from the chromatofocusing column were analyzed by native IEF electrophoresis. Protein bands were electroblotted onto PVDF membrane for N-terminal sequence analysis or extracted from IEF gel and rerun on SDS-PAGE to determine the subunit composition of each GST dimer. CL 2, CL 3, and CL 5 can form homodimers, whereas CL 1 and CL 4 exist only as CL 1-2 and CL 3-4 heterodimers. CL 2 and CL 5 have N-terminal amino acid sequences homologous to rat liver Yb and Ya GSTs, respectively. CL 1 has a unique N-terminal sequence that is not homologous to any known GSTs.     

Induction of glutathione-S-transferase in the Northern quahog Mercenaria mercenaria after exposure to the polychlorinated biphenyl (PCB) mixture Aroclor 1248

The glutathione-S-transferases (GSTs) from the Northern quahog (Mercenaria mercenaria) were examined after an injection of a polychlorinated biphenyl (PCB) mixture, Aroclor 1248, to a concentration of ~50 ppm. Enzymatic analysis indicated a fourfold increase in the GST activity of quahogs injected with PCBs compared with that of the control. An electrophoretic analysis of the GST from the PCB-exposed quahogs showed a 1.5-fold increase in the concentration over that of the control. Purification of the GST on a glutathione affinity column yielded a glutathione binding protein, in addition to the GSTs. However, the amount of the glutathione binding protein in the PCB-injected quahogs was found to decrease by ~50% in comparison to the glutathione binding protein in the control quahogs.     

Identification, characterization, and crystal structure of the Omega class glutathione transferases

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.