Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection Effect of the denaturation on the determination of thiolic proteins

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol dm⁻³ urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO₃ in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg²⁺, present in solution as Hg²⁺-urea complex, is selectively detected by AFS in a Ar/H₂ miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10⁻¹⁰-10⁻¹² mol dm⁻³, depending on the considered protein, with linear calibration curves spanning over four decades of concentration.     

Study of the disulfide reduction of denatured proteins by liquid chromatography coupled with on-line cold-vapor-generation atomic-fluorescence spectrometry (LC–CVGAFS)

Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomic-fluorescence spectrometry (HIC–CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA), -lactalbumin (-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the –SH groups of proteins with p-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. Hg^II, derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H₂ miniaturized flame after sodium borohydride (NaBH₄) reduction to Hg°. The yield of the reduction was studied as a function of reductant concentration, reduction time (t_red), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and for t_red were between 1 and 100 mmol L^–1 and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S–S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S–S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1–5×10^–12 mol L^–1 (10–30 pg), depending on the protein considered.     

Analysis of metallothioneins by means of capillary electrophoresis coupled to electrospray mass spectrometry with sheathless interfacing

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.     

Determination of S-nitrosoglutathione and other nitrosothiols by p-hydroxymercurybenzoate derivatization and reverse phase chromatography coupled with chemical vapor generation atomic fluorescence detection

S-Nitrosoglutathione (GSNO) reacts with the organic mercurial probe, p-hydroxymercury benzoate (PHMB, HO-Hg-(C₆H₄)-COO⁻Na⁺) giving the complex GS-Hg(C₆H₄)COOH (GS-PHMB). This reaction has been studied by UV measurements at 334 nm also in the presence of ascorbic acid and the product of reaction, the GS-PHMB complex, characterized by Electrospray Ionization Mass Spectrometry (ESI-MS) and by Reversed Phase Chromatography (RPLC) coupled on-line and sequentially with a UV-visible diode array detector (DAD) followed by a cold vapor generation atomic fluorescence spectrometer (CVGAFS). The simultaneous presence of PHMB and ascorbate produced a synergistic effect on GSNO decomposition rate that can be observed only above a given concentration threshold of ascorbate (ascorbate/GSNO molar ratio ≥ 180). The results indicated that the formation of GS-PHMB, both in the presence and the absence of ascorbic acid, does not involve the formation of free thiolic species but it takes place through a more complex mechanism. The PHMB derivatives of GSH and GSNO obtained by the present method were found to be identical by ESI-MS. GSSG did not interfere because it was not reduced and derivatized to GS-PHMB. Once complexed by the alkylating agent N-ethylmaleimide (NEM), GSH did not interfere with the derivatization reaction. This ensured a good selectivity of the developed PHMB derivatization system for RSNO determination. Thus, we have optimized the operating conditions for the selective reaction of PHMB with GSNO and other nitrosothiols (RSNOs) in order to determinate RSNOs in human plasma. LODc for RSNOs in plasma ultrafiltrate was 30 nM (injected concentration, 50 μL loop), the DLR ranged between 0.08 and 50 μM and the CV% was 6.5% at 300 nM concentration level. Reduced and oxidized thiols spiked to plasma did not interfere with the measurement of RSNOs. We found that the sampling procedure was critical for the recovery of endogenous and spiked RSNOs. The ultrafiltrate samples of plasma of 8 healthy humans contained 1460 ± 310 CysNO, 1000 ± 330 nM HCysNO and 320 ± 60 nM GSNO if blood was sampled in a mixture NEM/ethylendiaminotetracetic acid (EDTA)/serine-borate complex (SBC), where serine-borate complex is a potent inhibitor of γ-glutamyltransferase, an enzyme involved in the conversion of GSNO into CysGlyNO. In the absence of SBC during the sampling of blood GSNO concentration found in the ultrafiltrate was lower (at level of the determination limit in plasma ultrafiltrate, i.e. 75 nM) and the peak of CysGlyNO appeared, which corresponded to a concentration of 200 ± 60 nM (N = 4 blood samples).     

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

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.     

Determination of hydrogen sulfide and volatile thiols in air samples by mercury probe derivatization coupled with liquid chromatography-atomic fluorescence spectrometry

A new procedure is proposed for the sampling and storage of hydrogen sulphide (H₂S) and volatile thiols (methanethiol or methyl mercaptan, ethanethiol and propanethiol) for their determination by liquid chromatography. The sampling procedure is based on the trapping/pre-concentration of the analytes in alkaline aqueous solution containing an organic mercurial probe p-hydroxymercurybenzoate, HO-Hg-C₆H₄-COO⁻ (PHMB), where they are derivatized to stable PHMB complexes based on mercury-sulfur covalent bonds. PHMB complexes are separated on a C₁₈ reverse phase column, allowing their determination by liquid chromatography coupled with sequential non-selective UV-vis (DAD) and mercury specific (chemical vapor generation atomic fluorescence spectrometry, CVGAFS) on-line detectors. PHMB complexes, S(PHMB)₂CH₃S-PHMB, C₂H₅S-PHMB and C₃H₇S-PHMB, are stable alt least for 12 h at room temperature and for 3 months if stored frozen (−20 °C).The best analytical figures of merits in the optimized conditions were obtained by CVGAFS detection, with detection limits (LODc) of 9.7 μg L⁻¹ for H₂S, 13.7 μg L⁻¹ for CH₃SH, 17.7 μg L⁻¹ for C₂H₅SH and 21.7 μg L⁻¹ for C₃H₇SH in the trapping solution in form of RS-PHMB complexes, the relative standard deviation (R.S.D.) ranging between 1.0 and 1.5%, and a linear dynamic range (LDR) between 10 and 9700 μg L⁻¹. Conventional UV absorbance detectors tuned at 254 nm can be employed as well with comparable R.S.D. and LDR, but with LODc one order of magnitude higher than AFS detector and lower specificity. The sampling procedure followed by LC-DAD-CVGAFS analysis has been validated, as example, for H₂S determination by a certified gas permeation tube as a source of 3.071 ± 0.154 μg min⁻¹ of H₂S, giving a recovery of 99.8 ± 7% and it has been applied to the determination of sulfur compounds in real gas samples (biogas and the air of a plant for fractional distillation of crude oil).     

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     

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.     

Spectrofluorimetric study of the beta-cyclodextrin:vitamin K3 complex and determination of vitamin K3

Vitamin K(3) (menadione) is an oil-soluble vitamin and not naturally fluorescent but yields fluorescence when it is reduced. However, it is possible to yield a fluorescent derivative in the region of 407 nm in aqueous medium when complexed to beta-cyclodextrin (CD). A 1:1 stoichiometric ratio and a formation constant of 373+/-34 l mol(-1) were obtained for the binary inclusion complex between menadione and beta-CD. The measurements were performed at pH 6.2 adjusted by adding 0.1 mol l(-1) citrate buffer solution and 6.4 x 10(-3) mol l(-1) of beta-CD concentration. The calibration graph was linear over the range 0.1-2.0 mg l(-1) with a repeatability of 2.2%; the detection limit was 0.022 mg l(-1) and the limit of quantification limit was 0.073 mg l(-1). The procedure was applied to pharmaceutical formulations.     

Characterisation of human metallothioneins from foetal liver and adult kidney using differential pulse polarography

The electrochemical behaviour of four human metallothioneins (MT) from foetal liver and adult kidney with respect to solution pH as well as the influence of the addition of cadmium and/or zinc were studied using differential pulse polarography. The complexation equilibrium of dissociation and the formation from their metal depleted form was investigated, going from basic to acid solution and vice versa. The stability of these human MTs with respect to changes in the pH of the solution is low. In fact, the complexation equilibrium is not reversible and consequently, the protein, at acid pH, is denatured and the metal binding capacity to complex the cations through the thiol groups is lost. In the case of some of these MTs two different electrochemical responses due to the reduction of complexed Cd(II), as well as to the Zn(II) complexes were distinguished. The predominance of each of these two species for both cations seems to depend on the total concentration, on the ratio between zinc and cadmium concentrations and on the solution pH. In each of the molecules studied, the addition of zinc provokes the transformation of the peak attributed to the Cd-Thionein complex CdT, initially found in the native MT, into another form, CdT′, having different characteristics to the initial one. The MTs are able to incorporate the added metal ions into their molecule. This implies a new reorganisation of the initial molecule in which both cadmium and zinc complexes are involved. The apparent stability constants of the Cd-MT, Zn-MT and Hg-MT complexes were estimated at different pH values.     

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。     

Cobalt determination with FI-FAAS after on-line sorbent preconcentration using 1-nitroso-2-naphthol

The suitability of 1-nitroso-2-naphthol (NN) as a complexing agent for on-line preconcentration of cobalt using C(18) microcolumn with FI-FAAS system has been tested. Various parameters affecting the complex formation and its elution were optimized. Reagent solution (2.5x10(-3) mol l(-1)) and aqueous sample solution acidified with 0.1% (v/v) nitric acid were on-line mixed in a reaction coil set at 65+/-1 degrees C and flowed through the microcolumn for 30 s. The pH of the mixed solution was adjusted to 3 approximately 4 by adding HNO(3) (1 mol l(-1)) or NaOH (1 mol l(-1)) in the reagent solution. The adsorbed complexes in the microcolumn were eluted with ethanol (acidified to 1% nitric acid) in 10 s into the nebulizer of FAAS. A good precision (1.6% for 100 mug l(-1) Co(II), n=10), high enrichment factor 17.2, with detection limit (3sigma) 3.2 mug l(-1), and sample throughput (90 h(-1)) were obtained. The method was applied on the certified reference materials (CRMs) i.e. NBS-362 and NBS-364 (special low alloy steel), for the determination of cobalt and the results were in good agreement with the certified values.     

Adsorption and thermodynamic characteristics of Hg(II)-SCN complex onto polyurethane foam

The sorption of Hg(II) in the presence of sodium thiocyanate solution onto polyurethane (PUR) foam, an excellent sorbent, has been investigated in detail. Maximum sorption of Hg(II) is achieved from 0.1 M hydrochloric acid solution containing 7.5x10(-2) M sodium thiocyanate in 5 min. The sorption data followed both Freundlich and Langmuir adsorption isotherms. The Freundlich constants 1/n and sorption capacity, C(m), are evaluated to be 0.44+/-0.02 and (3.86+/-0.89)x10(-3) mol g(-1). The saturation capacity and adsorption constant derived from Langmuir isotherm are (6.88+/-0.28)x10(-5) mol g(-1) and (5.6+/-0.37)x10(4) dm(3) mol(-1) respectively. The mean free energy (E) of Hg(II)-SCN sorption onto PUR foam computed from D-R isotherm is 12.4+/-0.3 kJ mol(-1) indicating ion-exchange type mechanism of chemisorption. The variation of sorption with temperature yields thermodynamic parameters of DeltaH=-30.7+/-1.2 kJ mol(-1), DeltaS=-70.1+/-4.1 J mol(-1) K(-1) and DeltaG=-9.86+/-0.77 kJ mol(-1) at 298 K. The negative value of enthalpy and free energy reflects the exothermic and spontaneous nature of sorption. On the basis of the sorption data, sorption mechanism has been proposed.     

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.     

Temperature and pressure dependent rate coefficients for the reaction of Hg with Br and the reaction of Br with Br: a pulsed laser photolysis-pulsed laser induced fluorescence study

A pulsed laser photolysis-pulsed laser induced fluorescence technique has been employed to study the recombination of mercury and bromine atoms, Hg + Br + M --> HgBr + M (1) and the self-reaction of bromine atoms, Br + Br + M --> Br2 + M (2). Rate coefficients were determined as a function of pressure (200-600 Torr) and temperature (243-293 K) in nitrogen buffer gas and as a function of pressure (200-600 Torr) in helium buffer gas at room temperature. For reaction 1, kinetic measurements were performed under conditions in which bromine atoms were the reactant in excess concentration while simultaneously monitoring the concentration of both mercury and bromine. A temperature dependent expression of (1.46 +/- 0.34) x 10(-32) x (T/298)(-(1.86+/-1.49)) cm6 molecule(-2) s(-1) was determined for the third-order recombination rate coefficient in nitrogen buffer gas. The effective second-order rate coefficient for reaction 1 under atmospheric conditions is a factor of 9 smaller than previously determined in a recently published relative rate study. For reaction 2 we obtain a temperature dependent expression of (4.31 +/- 0.21) x 10(-33) x (T/298)(-(2.77+/-0.30)) cm6 molecule(-2) s(-1) for the third-order recombination rate coefficient in nitrogen buffer gas. The rate coefficients are reported with a 2sigma error of precision only; however, due to the uncertainty in the determination of absolute bromine atom concentrations and other unidentified systematic errors we conservatively estimate an uncertainty of +/-50% in the rate coefficients. For both reactions the observed pressure, temperature and buffer gas dependencies are consistent with the expected behavior for three-body recombination.     

A high-performance liquid chromatography/tandem mass spectrometric screening method for eight synthetic corticosteroids in bovine feces and the simultaneous differentiation between dexamethasone and betamethasone

A screening method was developed to monitor the illegal use of synthetic corticosteroids in cattle. Diethyl ether extracts from spiked feces samples were cleaned-up by solid phase extraction followed by semipreparative reversed-phase chromatography (RPC). The fraction containing the corticosteroids was derivatized with ethoxyamine hydrochloride. The corresponding ethoximes were separated using silica-based C18 RPC and analyzed on-line in an ion trap mass spectrometer using atmospheric pressure positive chemical ionization. Ethoxime derivatives of dexamethasone and betamethasone were baseline resolved, allowing for the simultaneous mass spectrometric differentiation of both epimers in bovine feces by conventional non-chiral chromatography. At the lowest level tested (1 micro g/kg), corticosteroids (except triamcinolone) could be identified in compliance with the recent European criteria for residue identification. The quantitative performance of the method was best at residue levels > or = 2 micro g/kg.     

Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay

Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.     

Flotation-spectrophotometric determination of mercury in water samples using iodide and ferroin.

This paper describes a simple and highly selective method for separation, preconcentration and spectrophotometric determination of trace amounts of mercury. The method is based on the flotation of an ion-associate of HgI4(2-) and ferroin between aqueous and n-heptane interface at pH 5. The ion-associate was then separated and dissolved in acetonitrile to measure its absorbance. Quantitative flotation of the ion-associate was achieved when the volume of the water sample containing Hg(II) was varied over 50 - 800 ml. Beer's law was obeyed over the concentration range of 3.2 x 10(-8) - 9.5 x 10(-7) mol l(-1) with an apparent molar absorptivity of 1 x 10(6) l mol(-1) cm(-1) for a 500 ml aliquot of the water sample. The detection limit (n = 25) was 6.2 x 10(-9) mol l(-1), and the RSD (n = 5) for 3.19 x 10(-7) mol l(-1) of Hg(II) was 1.9%. A notable advantage of the method is that the determination of Hg(II) is free from the interference of the almost all cations and anions found in the environmental and waste water samples. The determination of Hg(II) in tap, synthetic waste, and seawater samples was carried out by the present method and a well-established method of extraction with dithizone. The results were satisfactorily comparable so that the applicability of the proposed method was confirmed in encountering with real samples.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonate-derivatized oligosaccharides by capillary electrophoresis-electrospray ionization quadrupole ion trap mass spectrometry.

Dextran was partially hydrolyzed with 0.1 mol/l HCl and the hydrolysate was derivatized with 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) by reductive amination. The derivatized-oligosaccharide mixture was separated by capillary electrophoresis (CE) in a buffer of 1% HAc-NH4OH, pH 3.4, and the separated components were detected on-line by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS) in the negative ion mode. A mass accuracy lower than 0.01% could be achieved and as low as 1.6 pmol of detxran octaose could be detected. ANTS-derivatized dextran oligosaccharide with a degree of polymerization (DP) lower than 6 produced both [M-H]- and [M-2H]2- ions, whereas those with a DP of 6 or higher than 6 produced only [M-2H]2- ion. As 1< or =DP< or =6, the percentage of [M-2H]2- ion in the total ions of [M-H]- and [M-2H]2- was found to be a linear function of the logarithmic DP. Molecular mass determination with ESI-QIT-MS strengthens the power of CE analysis of oligosaccharides.     

金属硫蛋白家族内的结构域拼接

金属硫蛋白(Metallothioneins,MTs)由结构独立且功能明显区别的β,α两个结构域组成。神经生长抑制因子(NeuronalGrowthInhibitoryFactor,GIF)双名金属硫蛋白-Ⅲ(MT-Ⅲ),是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白,而β－结构域为其功能结构域。为深入系统地研究MTs,尤其是GIF及其结构域的结构与功能,我们构建了金属硫蛋白家族内结构域拼接体βGIF-αMT-1(βⅢ-αⅠ)和βMT-1-αGIF(βⅠ-αⅢ):PCR扩增得各个结构域的cDNA序列,酶切后克隆入原核表达载体pGEX-4T-1,经发酵、诱导表达、亲和层析、凝血酶切和进一步纯化,得率约为80mg蛋白/L菌液。测其电泳行为、氨基酸组成、质谱、金属巯基含量等,证明得到了目的蛋白。紫外吸收图谱和圆二色性图谱显示,结构域拼接体拥有金属硫蛋白家族成员的特征金属巯基簇结构域,初步功能实验表明,βⅢ－αⅠ也具有抑制神经元生长的功能。