外源钙对汞胁迫下菹草(Potamogeton crispus L.)叶片抗氧化系统及脯氨酸代谢的调节效应

以组织培养的菹草无菌苗为实验材料,研究了外源Ca(NO3)2对汞(Hg)胁迫下菹草无菌苗抗氧化酶系统、可溶性蛋白、总抗氧化能力(T-AOC)、非蛋白巯基(NP-SH)、植物络合素(PCs)以及脯氨酸代谢的调节效应。结果表明:(1)Hg胁迫使菹草体内过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)及谷胱甘肽还原酶(GR)活性明显降低,但超氧化物歧化酶(SOD)及过氧化物酶(POD)的活性并没有明显变化。外源添加25 mmol·L-1Ca(NO3)2后,明显提升了SOD、POD、APX及GR的活性;(2)Hg胁迫使菹草体内可溶性蛋白含量及T-AOC大幅降低,并引起NP-SH和PCs大量积累。外添Ca(NO3)2则有效减缓可溶性蛋白含量及T-AOC的下降,显著降低NP-SH和PCs水平;(3)脯氨酸是植物体内重要的渗透调节物质,外源Ca添加后,脯氨酸代谢关键酶吡咯啉-5-羧酸合成酶(P5CS)及δ-鸟氨酸转氨酶(OAT)活性显著增加,脯氨酸含量显著高于对照组。以上结果表明,Hg胁迫打破了菹草无菌苗抗氧化酶系统的内在平衡,而外添Ca(NO3)2有效促进了可溶性蛋白合成,提高了抗氧化酶活性和抗氧化物质含量,使菹草无菌苗维持较高的总抗氧化能力,并通过调节脯氨酸代谢来减轻Hg胁迫对菹草的伤害,增强植物对重金属Hg的耐受能力。     

锌胁迫对桉树巯基物质含量及抗氧化酶活性的影响

通过桉树的水培实验,测定了不同浓度Zn胁迫下桉树的生物量,根和叶中Zn、巯基物质(非蛋白巯基,谷胱甘肽和植物络合素)和丙二醛(MDA)的含量,以及根中抗氧化酶的活性。从巯基物质含量和抗氧化酶活性的角度,探讨了桉树抵御Zn胁迫造成氧化性伤害的机制。实验结果表明,Zn浓度较低(20 mg/L和40 mg/L)时,根中MDA含量非显著性增加,抗氧化酶超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性上升,叶中巯基物质含量非显著性增加。Zn浓度较高(120 mg/L和140 mg/L)时,根中MDA含量显著性增加,SOD、POD和CAT的活性下降,叶中巯基物质含量显著性增加。因此,低浓度Zn胁迫时,桉树中巯基物质和抗氧化酶系统对氧化性伤害有较好的缓解作用;高浓度Zn胁迫时,大量过氧化物积累,桉树中巯基物质和抗氧化酶系统对氧化损伤的缓解作用降低。     

枯草芽孢杆菌β-甘露聚糖酶活性中心氨基酸的化学修饰

运用化学修饰方法对枯草芽孢杆菌β-甘露聚糖酶活性中心的结构进行了研究。结果表明,除了色氨酸和巯基(Cys)外,羧基(Asp/Glu)和丝氨酸残基亦是该酶活性必需氨基酸残基;尽管组氨酸残基对酶活性的维持有重要作用,但不位于酶的活性中心。     

原核表达hbFGF结构与功能优化的研究

野生型hbFGF蛋白可溶性与稳定性较低一直是困扰研发人员的难题。分析其氨基酸序列发现含有 4个游离巯基 ,其中第 78、96、1 0 1位半胱氨酸游离巯基可能直接影响其可溶性、稳定性及活性。对天然hbFGF进行定点突变 ,一组将Cys78、Cys96同时突变 ,另一组将Cys78、Cys96与Cys1 0 1进行联合突变 ,均改为丝氨酸密码子 ,然后克隆入原核表达载体pET 3c ,进行表达 ,结果发现两种突变蛋白的可溶性大幅度增加 ,稳定性明显上升 ,其中三点突变的效果更为显著 ,但蛋白质的活性明显下降。可以认为 78、96与 1 0 1位半胱氨酸的游离巯基同时参与了天然hbFGF多聚体的形成 ,是导致野生型hbFGF可溶性与稳定性较低的主要原因 ,Cys1 0 1还与维持hbFGF的活性直接有关     

一种新型贻贝足丝抗氧化蛋白的原核重组表达及功能

贻贝足丝及其足丝蛋白相关研究对于开发新型水下生物粘附剂具有重要的仿生学意义。足丝蛋白在其粘附过程中需要维持一定的还原态,而目前已报道的足丝抗氧化蛋白仅有MFP-6。此前在厚壳贻贝足丝中鉴定到一种新型的富含半胱氨酸和甘氨酸的足丝蛋白质,该蛋白质被命名为Cys/Gly-Rich-Protein（CGRP）,但是CGRP蛋白在足丝中的作用及机制尚不明确。为此,针对CGRP蛋白,在序列分析基础上,利用原核重组表达手段获得其重组蛋白质,采用2,2-联苯基-1-苦基肼基（2,2-diphenyl-1-picryl hydrazyl radical,DPPH）法检测CGRP重组蛋白经不同条件处理后的抗氧化活性。序列分析结果表明,CGRP蛋白含16.5%的半胱氨酸和10%的甘氨酸,其序列中含有两段半胱氨酸位置保守的重复序列,结构预测表明,其优势构象以无规卷曲为主。同源蛋白质搜索结果表明,CGRP蛋白在数据库中尚无高同源性蛋白质存在。通过密码子优化结合原核重组表达策略成功表达出CGRP重组蛋白,所获得的CGRP重组蛋白具有明显的抗氧化活性,且该活性在其半胱氨酸还原后显著增强(0.91±0.05 vs 0.71±0.11, P<0.01)而在半胱氨酸烷基化之后显著下降(0.08±0.03 vs 0.71±0.11, P<0.01),表明CGRP蛋白的抗氧化活性与其序列中半胱氨酸的自由巯基有关。本研究提示,CGRP蛋白是足丝中一种新的具有抗氧化功能的蛋白质,在足丝粘附过程中推测与MFP-6一起参与了富含多巴的足丝粘附蛋白的还原态维持,对贻贝足丝在固化和粘附过程中防止提前粘附具有重要意义。     

多年生黑麦草抗氧化酶和植物络合素对Cd2+胁迫的应答

采用水培方法研究了5 mg· L-1 Cd2+胁迫下,Cd在多年生黑麦草中的积累和Cd2+对多年生黑麦草抗氧化酶活性和植物络合素等巯基化合物浓度的影响.将具有3片展开叶的多年生黑麦草实生苗转至1/2霍格兰营养液中培养2周后,对其进行5 mg-L-1 Cd2+处理,分别在处理后的0、0.25、1、3、6d取样测定根系和叶片的Cd浓度、抗氧化酶活性和植物络合素等巯基化合物的浓度.结果表明,Cd2+处理多年生黑麦草6d后,根系中Cd浓度达到2.59 mg·g-1,叶片中Cd浓度达到0.24 mg·g-1,根中Cd向叶片的转运系数力0.093,叶中Cd的富集系数为48,多年生黑麦草属Cd高积累植物,具备在植物修复上应用的前景.Cd2+胁迫下,多年生黑麦草根叶中丙二醛(MDA)含量无显著变化,根中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性无显著变化,抗坏血酸过氧化物酶(APX)对Cd2+敏感,处理后6d活性较0d显著下降67.19％.Cd2+处理1d内,叶中SOD、APX、CAT活性显著降低.Cd2+处理后3d,叶中的抗氧化酶系统对叶中Cd浓度的升高做出了正反馈,SOD、APX、CAT的活性分别较处理后1d显著上升了14.19％、76,82％、99.26％,Cd2+处理时间延长至6d,SOD活性较处理后3d显著下降了18.58％,APX、CAT活性无显著变化.Cd2+处理后6d,多年生黑麦草根中半胱氨酸(Cys)、谷胱甘肽(GSH)、植物络合素2(PC2)、植物络合素3(PC3)、植物络合素4(PC4)、植物络合素5(PC5)和植物络合素6(PC6)浓度分别较处理0d提高了2.19、1.57、2.06、16.08、5.73、6.03和4.31倍,叶中Cys、GSH、PC2、PC3和PC4浓度分别较处理0d提高了0.69、3.21、1.64、5.73和0.27倍.根中PC3巯基比例最大,叶中GSH的巯基比例最大,二者是根、叶中巯基存在的主要形式.随着Cd2+处理时间的延长,根系和叶片中各巯基化合物的总巯基浓度显著升高,根系和叶片中植物络合素总巯基浓度与Cd浓度显著正相关.多年生黑麦草通过植物络合素等巯基化合物的快速合成降低了根叶中自由Cd2+的比例,保护了根叶中抗氧化酶的活性,间接维持了活性氧代谢的平衡.     

硫氧还蛋白与心血管疾病

硫氧还蛋白是细胞内最重要的二硫键还原酶,对维持细胞内蛋白质的还原状态并正常发挥功能着重要的作用,此外。硫氧还蛋白、硫氧还蛋白还原酶和硫氧还蛋白过氧化物酶组成了细胞内最重要的抗氧化系统之一,在对抗细胞的氧化应激上起着重要作用。心血管疾病是威胁人类健康的主要疾病,它与炎症反应和氧化应激有着密切的联系。文章将从硫氧还蛋白的抗氧化、抗炎、抗细胞凋亡,调控与炎症基因表达有关的核转录因子的转录活性,以及调节细胞内蛋白质的亚硝基化等诸多方面阐述硫氧还蛋白在防御心血管疾病方面可能具有的生物学功能。     

CYP19基因表达与芳香化酶活性调控因子的研究进展

芳香化酶是雌激素合成中的关键酶,催化睾酮和雄烯二酮转化为雌激素。本文在对芳香化酶的蛋白结构、基因特征和分布进行阐述的基础上,重点对编码该蛋白的CYP19基因的调控因子以及芳香化酶活性的调节进行探讨。CYP19基因的调控因子包括cAMP反应元件(CRE)、类固醇生成因子1/肾上腺4结合蛋白(SF-1/Ad4BP)、雌激素受体(ER)等顺式作用因子和TATA结合蛋白(TBP)、生长因子等反式作用因子。主要通过cAMP依赖性蛋白激酶信号通路在转录水平对其进行调节。而芳香化酶表达及其酶活性的调控因子主要集中于性类固醇激素和促性腺激素,此外还受到温度等外部因子等因素的调节。芳香化酶的调节对维持雌雄激素间作用的平衡、保证机体的正常生理功能具有重要意义。     

浓核病毒感染早期家蚕血液、中肠蛋白表达差异比较分析

为了探明在浓核病毒镇江株(BmDNV-ZJ)侵染早期,家蚕部分组织蛋白所产生的免疫抵抗性变化机制,本实验采用差异蛋白质组学技术研究分析了BmDNV-ZJ感染早期,家蚕的中肠、血液组织中特异性表达的差异蛋白.实验结果表明:在浓核病毒侵染初期,感受性家蚕的中肠组织受病毒感染而得到特异性表达的蛋白可能为丝氨酸蛋白酶抑制剂和巯基抗氧化酶蛋白,前者具有调控蛋白酶活性和细胞凋亡的功能,后者具有抗氧化的作用.血液组织受病毒感染诱导而产生的蛋白可能是丝裂原活化蛋白激酶和类抗氧化酶蛋白,前者具有调控细胞凋亡的功能,后者具有抗氧化、消除自由基作用.由于试验中所得的差异蛋白点很少,这表明在BmDNV-ZJ感染早期,蚕体对浓核病毒的感染而产生的反应很小.蚕体可通过被侵染的中肠组织(浓核病毒感染的靶部位)以及血液(免疫组织)共同产生一些抗氧化或调控细胞凋亡的酶蛋白等来抗击浓核病毒的侵染.     

硒在糖尿病中抗氧化的作用及研究进展

糖尿病(diabetes mellitus, DM)是一种慢性内分泌代谢性疾病，氧化应激的增加是糖尿病的一个重要因素，氧化应激也被认为是导致糖尿病病程变化的关键因素。硒作为一种功能性的微量元素，以硒蛋白的形式(如谷胱甘肽过氧化物酶、硫氧还蛋白氧化还原酶)体现其生理功能，硒蛋白作为抗氧化蛋白在糖尿病的氧化应激机制中发挥重要的调节作用，参与抗氧化的主要环节。本文主要介绍硒在糖尿病中抗氧化的相关作用，为硒在预防和治疗糖尿病中的应用提供理论基础。     

Characterization of Rhodobacter sphaeroides cytochrome c(2) proteins with altered heme attachment sites

In c-type cytochromes, heme is attached to the polypeptide via thioether linkages between vinyl groups on the tetrapyrrole ring and cysteine thiols in a CX(2)CH motif. To study the role of the heme-binding site in c-type cytochrome assembly and function, we generated amino acid changes in this region of Rhodobacter sphaeroides cytochrome c(2) ((15)Cys-Gln-Thr-Cys-His(19)). Amino acid substitutions at Cys(15), Cys(18), or His(19) produced mutant proteins that did not support growth via photosynthesis where this electron carrier is required. Many of these changes appeared to slow signal peptide removal, suggesting that heme attachment is coupled to processing of the c-type cytochrome precursor protein. Inserting an alanine between the cysteine ligands (CycA-Ins17A) did not significantly alter the behavior of this protein in vivo and in vitro, suggesting that the existence of 2 residues between cysteine thiols is not essential for heme attachment to a Class I c-type cytochrome like cytochrome c(2).     

Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10 mM and/or reaction time to below 5 min. Maximal removal of Cys activity was observed in tissue homogenates at 40 mM NEM within 1 min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.     

Regulation of cathepsin B activity by cysteine and related thiols.

We studied the mode of regulation of the activity of mature cathepsin B (CB) by L-cysteine and some related thiols. The activity of CB with Z-Arg-Arg-NHMec as substrate was gradually inhibited over a range of increasing concentration of Cys, Cys methyl ester (CysOMe), Cys ethyl ester (CysOEt), N-acetyl-Cys (N-AcCys) and 3-mercaptopropionic acid. However, the inhibition of CB peaked at a definite value of [Cys], [CysOMe], [CysOEt] and [N-AcCys] and was gradually reversed over a range of higher concentrations of Cys and its esters. The maximum inhibitory concentrations of Cys, CysOME, CysOEt and N-AcCys showed a positive relationship to the pKa(RSH) values of the thiols and those of CysOEt and Cys decreased with increasing pH. The capability of the thiols to overcome their own inhibitory effect on CB was dependent on the concentration of their thiolate anion (RS-). However, the preincubation-dilution experiments showed that Cys and N-AcCys did not interact with active CB via a covalent mode. The inhibition of CB by N-AcCys was competitive and could be reversed by CysOMe. This activity-recovering effect of CysOMe was concentration-dependent and obeyed the Michaelis-Menten saturation kinetics over a profound increase of [RS-]. CB reacting in an environment of concurrently decreasing [RS-] and increasing [RSH], which was achieved by means of carboxylesterase-catalyzed deesterification of CysOEt to Cys, was progressively inhibited. Cys and N-AcCys also inhibited the fragmentation of histone H4 by CB and their concentration-dependent inhibitory profiles were qualitatively similar to those observed with Z-Arg-Arg-NHMec. Taken together, the results indicate that the RSH form of Cys and related thiols inhibits the activity of CB while the RS- form of these thiols counteracts or reverses the inhibitory action of the RSH form. This previously unrecognized thiol-thiolate anion regulation mechanism might be involved in a dynamic regulation of CB activity in endosomes and lysosomes and at the sites of lysosome-driven pericellular proteolysis.     

Shear-induced disulfide bond formation regulates adhesion activity of von Willebrand factor

von Willebrand factor (VWF) is the largest multimeric adhesion ligand circulating in blood. Its adhesion activity is related to multimer size, with the ultra-large forms freshly released from the activated endothelial cells being most active, capable of spontaneously binding to platelets. In comparison, smaller plasma forms circulating in blood bind platelets only under high fluid shear stress or induced by modulators. The structure-function relationships that distinguish the two types of VWF multimers are not known. In this study, we demonstrate that some of the plasma VWF multimers contain surface-exposed free thiols. Physiological and pathological levels of shear stresses (50 and 100 dynes/cm(2)) promote the formation of disulfide bonds utilizing these free thiols. The shear-induced thiol-disulfide exchange increases VWF binding to platelets. The thiol-disulfide exchange involves some or all of nine cysteine residues (Cys(889), Cys(898), Cys(2448), Cys(2451), Cys(2490), Cys(2491), Cys(2453), Cys(2528), and Cys(2533)) in the D3 and C domains as determined by mass spectrometry of the tryptic VWF peptides. These results suggest that the thiol-disulfide state may serve as an important structural determinant of VWF adhesion activity and can be modified by fluid shear stress.     

Mapping the cysteine proteome: analysis of redox-sensing thiols

The cysteine (Cys) proteome includes 214,000 Cys with thiol and other forms. A relatively small subset functions in cell signaling, while a larger number coordinate cell functions in response to redox state. The former are redox-signaling thiols while the latter are defined as redox-sensing thiols. Bulk measurements are not very informative for systems biology because reactivity of thiols in proteins differs by seven orders of magnitude. Proteomic databases contain annotation of Cys, for example, disulfides and zinc fingers, but do not include quantitative information necessary to develop functional models. Complementary databases and Cys proteome maps are needed to describe thiol redox circuits and connect these to functional redox-dependent pathways. This article summarizes progress in quantitative redox proteomics to develop such maps.     

Redox properties of the tissue factor Cys186-Cys209 disulfide bond

TF (tissue factor) is a transmembrane cofactor that initiates blood coagulation in mammals by binding Factor VIIa to activate Factors X and IX. The cofactor can reside in a cryptic configuration on primary cells and de-encryption may involve a redox change in the C-terminal domain Cys(186)-Cys(209) disulfide bond. The redox potential of the bond, the spacing of the reduced cysteine thiols and their oxidation by TF activators was investigated to test the involvement of the dithiol/disulfide in TF activation. A standard redox potential of -278 mV was determined for the Cys(186)-Cys(209) disulfide of recombinant soluble TF. Notably, ablating the N-terminal domain Cys(49)-Cys(57) disulfide markedly increased the redox potential of the Cys(186)-Cys(209) bond, suggesting that the N-terminal bond may be involved in the regulation of redox activity at the C-terminal bond. Using As(III) and dibromobimane as molecular rulers for closely spaced sulfur atoms, the reduced Cys(186) and Cys(209) sulfurs were found to be within 3-6 ? (1 ?=0.1 nm) of each other, which is close enough to reform the disulfide bond. HgCl2 is a very efficient activator of cellular TF and activating concentrations of HgCl2-mediated oxidation of the reduced Cys(186) and Cys(209) thiols of soluble TF. Moreover, PAO (phenylarsonous acid), which cross-links two cysteine thiols that are in close proximity, and MMTS (methyl methanethiolsulfonate), at concentrations where it oxidizes closely spaced cysteine residues to a cystine residue, were efficient activators of cellular TF. These findings further support a role for Cys(186) and Cys(209) in TF activation.     

Mass spectrometric analysis of nitroxyl-mediated protein modification: comparison of products formed with free and protein-based cysteines

Although biologically active, nitroxyl (HNO) remains one of the most poorly studied NO(x). Protein-based thiols are suspected targets of HNO, forming either a disulfide or sulfinamide (RSONH2) through an N-hydroxysulfenamide (RSNHOH) addition product. Electrospray ionization mass spectrometry (ESI-MS) is used here to examine the products formed during incubation of thiol proteins with the HNO donor, Angeli's salt (AS; Na2N2O3). Only the disulfide, cystine, was formed in incubates of 15 mM free Cys with equimolar AS at pH 7.0-7.4. In contrast, the thiol proteins (120-180 microM), human calbindin D(28k) (HCalB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) gave four distinct types of derivatives in incubates containing 0.9-2.5 mM AS. Ions at M + n x 31 units were detected in the ESI mass spectra of intact HCalB (n = 1-5) and GAPDH (n = 2), indicating conversion of thiol groups on these proteins to RSONH2 (+31 units). An ion at M + 14 dominated the mass spectrum of BSA, and intramolecular sulfinamide cross-linking of Cys34 to one of its neighboring Lys or Arg residues would account for this mass increase. Low abundant M + 14 adducts were observed for HCalB, which additionally formed mixed disulfides when free Cys was present in the AS incubates. Cys149 and Cys153 formed an intramolecular disulfide in the AS/GAPDH incubates. Since AS also produces nitrite above pH 5 (HN2O3(-) --> HNO + NO2(-)), incubation with NaNO2 served to confirm that protein modification was HNO-mediated, and prior blocking with the thiol-specific reagent, N-ethylmaleimide, demonstrated that thiols are the targets of HNO. The results provide the first systematic characterization of HNO-mediated derivatization of protein thiols.     

Evidence for the participation of Cys558 and Cys559 at the active site of mercuric reductase

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH2 form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH2 has previously been reported to require only 2 electrons for reduction of the active site disulfide. We present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH2 actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4 degrees C and at 25 degrees C indicate that reduction of the active site occurs rapidly, as previously reported [Sahlman, L., & Lindskog, S. (1983) Biochem. Biophys. Res. Commun. 117, 231-237]; this is followed by a slower reduction of another redox group via reaction with the active site. Thiol titrations of denatured Eox and EH2 enzyme forms show that an additional disulfide is the group in communication with the active site. [14C]Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys558 and Cys559, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, Eox with Cys558 and Cys559 as thiols exhibits less than 50% of the fluorescence of Eox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.     

Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d⁷-ω-bromoacetonylquinolinium bromide (d⁷-BQB). BQB and d⁷-BQB both rapidly and selectively reacted with thiols in acidic medium within 3 min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d⁷-BQB, respectively. The BQB- and d⁷-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI–MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI–MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI–MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27 nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers.