Characterization of the metal composition of metallothionein isoforms using reversed-phase high-performance liquid chromatography with atomic absorption spectrophotometric detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate metallothionein (MT) isoforms and on-line atomic absorption spectrophotometric (AAS) detection was used to quantitatively determine their metal content. With this coupled system (HPLC-AAS), it was possible to determine the zinc, cadmium and copper content of individual horse kidney MT isoforms. When rabbit liver MT and the purified isoforms (MT-1 and MT-2) were subjected to RP-HPLC and the zinc-containing peaks of the MT sample to MT-1 or MT-2. HPLC-AAS was used to identify zinc-induced MT in heat-treated cytosol from turkey hen liver, thereby demonstrating its application to the analysis of crude tissue extracts. A standard curve was established using turkey liver MT for the quantitative determination of the zinc content of MT isoforms. There was excellent linear correlation between the micrograms of zinc bound to MT injected onto the column (ranging from 0.34 to 3.43 micrograms of MT-bond zinc) and the integrated peak area of the atomic absorbance for zinc. Using this standard curve, it was possible to quantitate the amount of MT-bound zinc in cytosol extracts of cultured turkey embryo hepatocytes exposed to varying levels of supplemental zinc in the culture medium.     

银诱导大鼠肝脏金属硫蛋白的提纯与鉴定

Ｗｉｓｔａｒ公鼠经腹腔注射ＡｇＮＯ３后可诱导肝脏合成ＭＴ。经匀浆、乙醇沉降、Ｓｅｐｈａｄｅｘ－７５、ＤＥＡＥ－５２两次柱层析，可得到两个亚型。原子吸收测定结果表明：该蛋白分别含７份Ａｇ、２份Ｚｎ和２份Ｃｕ，具有与Ｃｄ５Ｚｎ２－ＭＴ并不相同的二级、三级结构。进一步研究表明，蛋白中伴随Ｃｕ和Ｚｎ的含量与所用诱发剂的种类、数量均有关，且Ｃｕ和Ｚｎ（通过ＭＴ）具有某种微妙的联系。     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

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。     

兔肝金属硫蛋白亚型异构体的高效液相色谱分离与ESI-MS,MALDI-TOF-MS鉴定

由于金属硫蛋白(MT)基因的多态性,决定不同亚型的MT异构体的存在,MT亚型异构体的结构是MT功能研究的基础.通过离子交换柱可将MT分成MT-1和MT-2两个异构体,用不同条件的反相高效液相色谱(RP-HPLC)可将MT-1和MT-2分成不同的亚型异构体,并利用MALDI-TOFMS和LC-ESI-MS对比确定了它们的分子量.结果表明,兔肝MT在不同的pH条件下分离得到不同分子量的亚型异构体.在酸性条件下,MT-1可分为2个主要亚型异构体,分子量分别为6149.0和6244.5,而MT-2主要分为3个亚型异构体,分子量分别为6149.0,6244.0和6127.0.MT-1和MT-2有2个亚型异构体分子量相同的异构体存在.在酸性条件下,MT-1的2个异构体及MT-2分子量为6127的亚型异构体可稳定存在.     

金属硫蛋白的色谱/电喷雾电离质谱联用表征方法

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     

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris-HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM beta-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg(-1). In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.     

Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS

Pneumatically assisted electrospray mass spectrometry (ESMS) in the direct mode and as a chromatographic detection technique was developed for the characterization of horse kidney metallothionein isoforms. Direct analysis in an acidic medium showed the presence of three major and five minor isoforms, the molecular masses of which were determined. The presence of the major isoforms (two of which matched the molecular masses calculated according to the published sequences) was confirmed by complexation with Cd at pH 4.0 and the determination of the stoichiometry of the complexes formed. Reversed-phase chromatography of Cd7-MT complexes (pH 6.0) gave two signals corresponding to the MT-1A and MT-1B isoforms. A post-column acidification procedure was developed to eliminate the possibility of artefacts associated with the formation of mixed-metal (Cd, Zn) complexes during chromatography in neutral media, and to improve the accuracy of the determination of the molecular mass of MT isoforms.     

兔金属硫蛋白的分离与纯化

兔皮下注射ＣｄＣｌ２诱导金属硫蛋白（Ｍｅｔａｌｌｏｔｈｉｏｎｅｉｎ，简称ＭＴ），取肝脏匀浆，用乙醇─氯仿混合液萃取后经ＳｅｐｈｄｅｘＧ－５０柱分离，得到混合型ＭＴ．进一步用纤维素弱碱性阴离子交换树脂进行拆分，并在ＳｅｐｈａｄｅｘＧ－２５柱上脱盐，得到ＭＴ－１和ＭＴ－２两种亚型．经氨基酸组咸、凝胶电泳及ＨＰＬＣ柱层析等分析表明，所得的两种亚型具有高度的均一性．     

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

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.     

Speciation analysis of trace elements in the brains of individuals with Alzheimer's disease with special emphasis on metallothioneins

A speciation analysis of protein-bound elements in the cytosol of human brain was achieved by size exclusion chromatographical separation of the biomolecules and on-line detection of the metal profiles in the eluate by hyphenated inductively coupled plasma-mass spectrometry. Post-mortem samples from Alzheimer's disease brains and from brains of a control group were investigated to elucidate changes in the trace element distribution during the pathological process. Special attention was paid to the metallothioneins (MT) - cysteine-rich, metal-binding proteins of low molecular weight, existing in several isoforms. The isoform MT-3 is found especially in the brain and has a growth inhibition function on neurons. The MT peaks were identified in the element profiles. For this purpose, the metal binding capability and the heat stability of MT were taken into consideration. For verification, a comparison with pure MT-3 was carried out and further biochemical and analytical methods were applied to the fractions of the chromatographical run. A comparison between Alzheimer's disease and control brains showed a significant difference concerning the MT-1/-2 and MT-3 metal levels, leading to the assumption that there were oxidative processes having taken place in the Alzheimer's brain samples.     

An improved method for isolation and identification of Zn-metallothionein from cadmium-induced rat liver.

Zn-metallothioneins (MT-1 and MT-2) were isolated and purified from Wistar rat liver induced by subcutaneous injection with cadmium chloride over a short time. Instead of Sephadex G-50 and DEAE Sephadex A-50, new chromatographic media produced by Pharmacia, Sephacryl S-200, S-100 and DEAE Sepharose Fast Flow were used in the purification of metallothioneins. The time required for purification with the new method was only 1/3 that required with the usual method and had the same purification effect and rate of recovery. The number of mercapto groups measured with modified Ellman's reagent and cysteine as standard is 20 in MT molecules. Zn and Cd concentrations in each fraction were measured by single sweep polarography rather than atomic absorption spectrophotometry. MT-1 and MT-2 contained 6 gram atoms of zinc, but no cadmium. Purified MT-1 and MT-2 were shown by high performance liquid chromatographic analysis to be highly homogeneous and had an amino acid composition similar to that of Cd-MT.     

Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs’ polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.     

Characterization of denatured metallothioneins by reversed phase coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection

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.     

Characterization and quantification of metallothionein isoforms by capillary electrophoresis–inductively coupled plasma–isotope-dilution mass spectrometry

In a new approach to the characterization and quantification of metallothionein isoforms an on-line isotope-dilution method in combination with the coupling of capillary electrophoresis (CE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) is reported. Metallothionein (MT) isoforms are separated by CE and the elements Cu, Zn, Cd, and S are detected simultaneously by use of ICP-SFMS in the medium resolution mode. On-line isotope dilution is performed by continuous introduction of an isotopically enriched, species-unspecific spike solution after the separation step. MT from rabbit liver and a further purified MT-1 isoform were quantified by determination of sulfur, and the stoichiometric compositions of the metalloprotein complexes are characterized by determination of their sulfur-to-metal ratios.     

Hapten synthesis and enzyme-linked immunosorbent assay for phosmet residues: assay optimization and investigation of matrix effects from different food samples

In this study, a panel of haptens was synthesized for immunoconjugate preparation, and several haptens for heterologous tracer conjugates were also prepared. A highly sensitive polyclonal antibody against the organophosphorus insecticide phosmet was obtained and competitive direct enzyme-linked immunosorbent assays (cd-ELISA) for this pesticide were developed. In the cd-ELISA, the limit of detection (IC₁₅) was 0.6 μg kg⁻¹ and the sensitivity (IC₅₀) was 20 μg kg⁻¹. The suitability of the ELISA for pesticide quantification in peach, apple, orange juice, and apple juice was also studied. Good accuracy and precision were obtained with mean recoveries between 78% and 102.3% and mean coefficients of variation below 13.63%. Validation of the ELISA was conducted by high-performance liquid chromatography. The correlation between the data obtained using the microwell assay and the high-performance liquid chromatography was good (R ² = 0.9849). The developed immunoassay methods were suitable for the rapid quantitative or qualitative determination of phosmet in food samples.     

Quantification of chitinase and thaumatin-like proteins in grape juices and wines

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.     

Purification and Characterization of a Mannose Recognition Lectin from <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> (Tilapia Fish): Cytokine Production in Mice Splenocytes

The aim of this work was to purify and partially characterize a mannose recognition lectin from Nile tilapia (Oreochromis niloticus) serum, named OniL. OniL was isolated through precipitation with ammonium sulfate and affinity chromatography (Concanavalin A–Sepharose 4B). In addition, we evaluated carbohydrate specificity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) profiles, and in vitro immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production. The ammonium sulfate fraction F2 showed the highest specific hemagglutinating activity (331) and was applied to affinity matrix. Adsorbed proteins (OniL) were eluted with methyl-α-d-mannopyranoside. OniL, a 17-kDa protein by SDS–PAGE constituted by subunits of 11 and 6.6 kDa, showed highest affinity for methyl-α-d-mannopyranoside and d-mannose. Immunological assays, in vitro, showed that OniL did not show cytotoxicity against splenocytes, induced higher IFN-γ production and lower IL-10 as well as nitrite release. In conclusion, OniL lectin was successfully purified and showed a preferential Th1 response in mice splenocytes.     

Syntheses, Characterization and Anti-HIV Activity of Charge Transfer Heteropolycomplexes

IntroductionAcquired immunodeficiency syndrome(AIDS) is a fatal disease caused by human immun-odeficiency virus type 1 (HIV-l ). Although this kind of disease was fOund only about tenyears ago, it has attracted extensive attention because Of its epidemic speed and high deathrate. Now, scientists are, at an unprecedented speed, accumulating the relevant knowledge inexpectation of discovering the methods to prevent and cure the disease. Furthermore, manystudies have indicated that the patien…     

Application of high‐speed counter‐current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high‐speed counter‐current chromatography using a two‐phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high‐performance liquid chromatography with an Agilent ZORBAX® SB‐C18 column (4.6 mm × 150 mm, 5 μm). After separation by high‐speed counter‐current chromatography and high‐performance liquid chromatography, we obtained 3.5 mg of platetyolin A, 4.1 mg of platetyolin B, and 18.1 mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high‐performance liquid chromatography and the chemical structures were evaluated by high‐resolution electrospray ionization time‐of‐flight mass spectrometry and one‐ and two‐dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.