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1.
通过反相液相色谱(RPLC)与电喷雾电离质谱(ESI-MS)的联用技术,对镉诱导金属硫蛋白标准物质MT-1和MT-2的结构进行表征分析。采用Vydac C8 反相色谱柱(250 mm×2.1 mm i.d., 5 μm, 30 nm),流动相A为pH 6.0的5 mmol/L乙酸铵水溶液,流动相B为pH 6.0的5 mmol/L乙酸铵的甲醇-水(体积比为1∶1)溶液,流动相流速为0.20 mL/min,在40 min内流动相B的体积分数从10%增加到37.5%进行梯度洗脱。分别用紫外(UV)和ESI-M  相似文献   

2.
The present study establishes a method for the separation and characterization of rabbit liver metallothionein (MT) subisoforms by capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry (CE-ESI-TOF-MS) via a sheath-flow interface. Directly coupled-CE-MS enables the extraction of specific molecular weight information and thereby facilitates the identification of peaks when no reference materials are available, as in the case of MT subisoforms. The analysis described here revealed the presence of the apothioneins MT-1a, MT-2d, and MT-2e, belonging to MT-I sample, and MT-2a, MT-2b, and MT-2c, belonging to MT-II. Several non-N-acetylated forms were also detected as traces appearing with their respective acetylated forms in both samples. Similar results were found when MALDI-TOF experiments were performed, identifying all the sequenced rabbit liver MTs as apo-MT-forms, as in the CE-ESI-MS coupling.  相似文献   

3.
通过凝胶色谱(SEC)及反相液相色谱(RPC)分别与电感耦合等离子体质谱(ICP-MS)和电喷雾电离质谱(ESI-MS)联用的技术,对灌喂HgCl2大鼠的肾组织中诱导的多金属结合金属硫蛋白(MT)的结构进行表征分析。通过SEC-ICP-MS曲线上的金属信号及紫外检测的MT光谱吸收特征,可确认组织提取液中MT在色谱曲线上的位置。与对照组相比,灌喂HgCl2大鼠肾中Hg-MT的诱导量显著增加,与此同时,Cu-MT在染毒组的诱导量也相应增加,说明MT在染汞大鼠肾脏中起着对汞的解毒和对铜的调节作用。MT样品提取物经葡聚糖凝胶(G-75)柱分离纯化并用透析法脱盐后,用细内径反相色谱柱分离,进行梯度洗脱,并与ESI-MS联用,可获得总离子流色谱图及与色谱峰对应的质谱图,通过对质谱信号的解析及参考脱金属MT构型的有关报道,对汞诱导大鼠肾组织MT的不同亚型和次亚型分子结构进行推断。结果证明,在灌喂HgCl2大鼠的肾组织中存在着一系列汞结合的MT形态。  相似文献   

4.
A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.  相似文献   

5.
A new analytical hyphenated technique is proposed for determination and characterization of thiolic proteins, based on reverse phase chromatography (RPC) coupled on-line with cold vapour generation atomic fluorescence spectrometry (CVGAFS). Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol l(-1) urea and p-hydroxymercurybenzoate (PHMB). The derivatized proteins are separated on a C4 Vydac Reverse Phase column. Post-column on-line reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of both the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury, also in the presence of methanol in the RPC eluent phase. Hg(II) is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg degrees. Under optimized conditions, on-line bromine treatment gives a 98+/-2% recovery of both free and protein-complexed PHMB. The effect of methanol on the sensitivity of Hg(II) detection was studied and controlled. RPC-CVGAFS system has been applied to the analysis of metallothioneins from rabbit liver (MT(RL)) standard solutions, and their commercial isoforms MT-1 and MT-2. The analysis of denatured, PHMB-complexed MTs allowed the determination of the number of thiolic groups complexed by PHMB. It was found that MTs from rabbit liver have 10.0+/-0.3 (MT-1) and 6.7+/-0.3 (MT-2 and MT(RL)) -SH groups complexed by PHMB. The detection limit (LODc) for PHMB in 95% methanol in the optimized conditions was about 9.3 x 10(-9) mol l(-1) and for the denatured MTs LODc was about 8.6 x 10(-10) mol l(-1), taking into account an approximate complexating ratio PHMB:MTs of 7:1.  相似文献   

6.
蛋白质数据库 (ProteinDataBank,PDB)中有大鼠及几个其它种属动物的金属硫蛋白的结构 ,但其中只有大鼠金属硫蛋白亚型Ⅱ的晶体结构是完整的单体[1],核磁共振溶液构象均只是金属硫蛋白被蛋白酶降解后的单一α或 β结构域.从大鼠金属硫蛋白亚型Ⅱ的晶体结构的堆积形式上看 ,二聚体可能是晶体生长过程中最小的堆积单元 ,因此Robbins和Stout等人认为 ,大鼠金属硫蛋白亚型Ⅱ分子在溶液中也是以二聚体形式存在[2].在晶体生长过程中 ,蛋白质在溶液中的构象会直接影响晶体的堆积.因此 ,对于大鼠金属硫蛋白…  相似文献   

7.
《中国化学快报》2023,34(1):107262
The transformation of quantum dots (QDs) by organisms has attracted broad attention but remains unclear. Understanding of the metabolites helps to reveal the transformation pathway of QDs. Cd containing-metallothionein (MT) are the main species formed by Cd released from CdSe QDs in HepG2 cells, while speciation analysis of Cd containing MTs remains a challenge because MTs has several subisoforms and can bind with several metals. Herein, we built a hyphenated platform for speciation analysis of QDs in HepG2 cells after treatment with CdSe/ZnS QDs. The Cd-containing MTs were separated in reversed phase high performance liquid chromatography (RP-HPLC) and subsequently online detected by inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) parallelly. Four groups of Cd-containing metabolites were found by detecting Cd in ICP-MS. Their structures were identified in ESI-Q-TOF-MS and further confirmed with standards of four subisoforms of MT, including N-terminal acetylation MT2a, N-terminal acetylation MT1e, N-terminal acetylation MT1g and MT1m. Each group of them contains various stoichiometry of Cd/Zn. The metabolites of QDs remain same while the concentrations of each metabolite and its stoichiometry of Cd/Zn vary for different incubation concentration/time. This work provides a new parallel hyphenation technique of HPLC-ICP-MS/ESI-MS with high separation resolution and powerful detection ability, and the obtained results provide detailed metabolism information of QDs in HepG2 cells after treatment of CdSe/ZnS QDs, contributing to deep exploration of the functional mechanisms of QDs in organisms.  相似文献   

8.
A rapid method is described for the determination of dietary cadmium-induced metallothioneins (MTs) in rabbit kidneys by anion-exchange high-performance liquid chromatography. Rabbit kidney MT-I and MT-II were eluted at ca. 15.0 and 18.8 min, respectively, from a DEAE-5PW anion-exchange column with a Tris-HCl buffer (0.01-0.25 M, pH 8.6) and detected by ultraviolet absorbance at 254 nm. A standard calibration curve was constructed using purified standard MT isoforms, which demonstrated an excellent linear correlation between UV absorbance peak heights and the amounts of MT isoforms. Feeding a dose of cadmium for some days resulted in an increase in MT concentrations in rabbit kidneys, but not in the livers. The cadmium concentrations in MT-I and MT-II elutions were determined by graphite furnace atomic absorption spectrometry. MT-I and MT-II showed some differences associated with the oral intake of cadmium. Dietary cadmium also caused zinc to accumulate in kidneys to some extent. The effects of dietary oleic acid on the synthesis of MTs were also studied. Based on the method of standard additions, the recovery of MTs exceeded 93% and replicated injection of samples yielded a relative standard deviation of 2.4% at an MT level of 280 micrograms/g.  相似文献   

9.
卢素格  沈金灿  庄峙厦  王小如 《色谱》2005,23(2):164-167
建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。  相似文献   

10.
A series of cationic oligomeric surfactants (quaternary dodecyldimethylammonium ions with two, three, or four chains connected by an ethylene spacer at the headgroup level, abbreviated as dimer, trimer, and tetramer) were synthesized and characterized. The influence of the degree of oligomerization on their polymorphic and mesomorphic properties was investigated by means of X-ray diffraction, polarizing optical microscopy, thermogravimetry, and differential scanning calorimetry. All compounds display layered arrangements with interdigitated dodecyl chains. The increase in the degree of oligomerization increases the interlayer distance and decreases the ordering in the solid phase; whereas the dimer sample is fully crystalline with well-developed 3D ordering and the trimer and tetramer crystallize as highly ordered crystal smectic phases. The number of thermal phase transitions and sequence of phases are markedly affected by the number of dodecyl chains. Anhydrous samples exhibit polymorphism and thermotropic mesomorphism of the smectic type, with the exception of the tetramer that displays only transitions at higher temperature associated with decomposition and melting. All hydrated compounds form lyotropic mesophases showing reversible phase transitions upon heating and cooling. The sequence of liquid-crystalline phases for the dimer, typical of concentrated ionic surfactant systems, comprises a hexagonal phase at lower temperatures and a smectic phase at higher temperatures. In contrast, the trimer and tetramer reveal textures of the hexagonal phase.  相似文献   

11.
Capillary electrophoresis (CE) coupled to electrospray mass spectrometry via sheathless interfacing has been applied to the analysis of mammalian metallothionein (MT) extracts. In a rabbit-liver extract, four (MT-2C, MT-2A, MT-2D and MT-2E) out of six known MT sub-isoforms were unambiguously identified under three CE-resolved peaks. A fourth peak was found to contain MT-1A and/or MT-2B, whose molecular masses differ by only 1 Da. Traces of non-N-acetylated MT-2D and MT-2E were observed in a fifth, minor peak. In a rat-liver extract, both MT-1 and MT-2 were resolved and identified. Non-N-acetylated MT-2 was also identified in a resolved, minor peak. Minimum detectable amounts of MTs have been estimated to be approximately 0.6 fmol per sub-isoform.  相似文献   

12.
Recent developments in the coupling of highly selective separation techniques such as capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) to element-specific and molecule-specific detectors, such as inductively-coupled plasma mass spectrometry (ICP-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) for the characterization and quantification of metallothioneins (MTs) are critically reviewed and discussed. This review gives an update based on the literature over the last five years. The coupling of CE to ICP-MS is especially highlighted. As a result of progress in new interface technologies for CE-ICP-MS, research topics presented in the literature are changing from "the characterization of interfaces by metallothioneins" to the "characterization of metallothioneins by CE-ICP-MS". New applications of CE-ICP-MS to the analysis of MTs in real samples are summarized. The potential of the on-line isotope dilution technique for the quantification of MTs and for the determination of the stoichiometric composition of metalloprotein complexes is discussed. Furthermore, a selection of relevant papers dealing with HPLC-ICP-MS for MT analysis are summarized and compared to those dealing with CE-ICP-MS. In particular, the use of size-exclusion (SE)-HPLC as a preliminary separation step for metallothioneins in real samples prior to further chromatographic or electrophoretic separations is considered. Additionally, the application of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) for the identification of metallothionein isoforms following electrophoretic or chromatographic separation is discussed.  相似文献   

13.
Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis.  相似文献   

14.
The complexing properties of the sub-isoforms of several mammalian Zn-containing metallothioneins (ZnMT) from different origins (rat, rabbit and human foetal livers) have been investigated by capillary zone electrophoresis coupled to diode array detection (CZE-DAD). MTs were submitted to three different experiments: gradual additions of Cd to MT at pH 8.0, variation of the pH and addition of Cd followed by a variation of the pH. On the whole there seems to be no displacement of the zinc originally present in the MT, but incorporation of Cd in the molecule in order to reach saturation. Furthermore, the pH variations allow one to conclude a certain reversibility of the system. All these results confirm those previously obtained by differential pulse polarography (DPP). The procedure used in this work shows that the sub-isoforms of an MT may exhibit variable complexing properties. This is particularly well illustrated in the case of rat liver MT-2. Much likely there are different mixed complexes of Cd and Zn in each sub-isoform, forming various metalloforms. This work illustrates a different and original use of CZE-DAD for studying the complexing properties of metal-binding proteins.  相似文献   

15.
The dimer, trimer, and tetramer of 1,11-dodecadiyne, HC?C? (CH2)8? C?CH, were synthesized. The solid-state polymerization of the dimer was investigated by infrared (IR) spectroscopy. IR bands due to the diacetylene moiety were identified through the comparison of the IR spectra of the dimer, trimer, and tetramer. The dimer was found to have two polymorphs, melt-crystallized and solution-crystallized. Both of the polymorphs undergo solid-state polymerization by exposure to γ-ray or UV irradiation. The former has higher polymerizability for the diacetylene moiety than the latter. The solid-state polymerization of the terminal acetylene group was not observed. It is shown that the previously reported dimer structure in which both the diacetylene and terminal acetylene groups are polymerized to form an inherently electrically conducting polymer is incorrect. © 1995 John Wiley & Sons, Inc.  相似文献   

16.
The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.  相似文献   

17.
《Electroanalysis》2006,18(21):2099-2105
Nafion‐coated bismuth film electrodes (NCBiFEs) were used to investigate the redox behavior of and metal release from rabbit liver metallothioneins (MTs) in an acetate buffer. Owing to the permselective exchange between positively charged MT molecules and cations in Nafion and the absence of detectable MT adsorption at bismuth surface, a diffusion‐controlled Nernstian redox wave of MTs (Epc=?0.869 V) was observed for the first time. The Nernstian behavior of the diffusing MTs is in contrast to the voltammetric responses of MTs at thin mercury films or Nafion‐coated mercury film electrodes, which either result in the replacement of the metals originally present in MTs by mercury or lead to a noticeable MT adsorption. By avoiding these undesirable features, the NCBiFE provides an excellent milieu for voltammetric studies of different types of MTs and related isoforms, paving the way for studying the redox‐modulated metal transfer of MTs and understanding the biological role of MTs in heavy metal detoxification and essential metal regulations.  相似文献   

18.
A study on electrochemical characterisation of three isoforms of human foetal liver Zn-metallothioneins, labelled MT-0, MT-1 and MT-2, has been performed by using differential pulse polarography (DPP). Two different peaks, attributed to two different Zn complexes with metallothioneins, have been detected. The electrochemical behaviour is similar for the three studied isoforms. Studies on the addition of Cd(2+) and Zn(2+) as well as studies as a function of pH have been carried out. The association and dissociation equilibria of metal ions with MTs are reversible in the studied pH range. The behaviour of Zn complexes in human foetal liver Zn-metallothioneins is comparable to the Cd complexes obtained using other mammalian Cd, Zn-metallothioneins, particularly as a function of pH.  相似文献   

19.
Syndiotactic poly(methyl methacrylate) (st-PMMA) macromonomer having methacryloyl end group was prepared from st-PMMA living anion and separated into uniform macromonomers by means of supercritical fluid chromatography. A uniform macromonomer with the degree of polymerization of 32 was polymerized radically in benzene at 60°C. The uniform dimer, trimer and tetramer of the uniform macromonomer were isolated from the polymerization product by means of gel-permeation chromatography (GPC). The intrinsic viscosity ([η]) in tetrahydrofuran of these uniform comblike polymers was determined by GPC/differential viscometric analysis. The plot of logarithmic [η] against logarithmic molecular weight indicated that the trimer and tetramer assume a little shrinking molecular shape as compared with the unimer and dimer.  相似文献   

20.
The structure, energetics, and vibrational spectra of the (HXeCN)2 dimer were investigated at the CCSD(T), MP2 and B3LYP levels. Such properties of the (HXeCN)3 trimer and (HXeCN)4 tetramer were investigated at the B3LYP level. The dimer, trimer, and tetramer were predicted to have a C2h, C2v, and D2d structure, respectively. In all of these oligomers, the N?Xe intermonomeric interaction is the most important one for holding the monomers together. Included with the ZPVE and BSSE, the stabilization energy of the dimer is 12.36 kcal/mol at the CCSD(T) level, while those of the dimer, trimer, and tetramer are 10.42, 18.23, and 31.34 kcal/mol, respectively, at the B3LYP level. At the B3LYP level, with respect to those of the isolated monomer, the C-Xe and Xe-H asymmetric stretching frequencies are shifted by -11.2 and +128.0 cm(-1) for the dimer, -51.6, +220.7 and -11.5, +96.6 cm(-1) for the trimer, and -14.1 and +201.8 cm(-1) for the tetramer.  相似文献   

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