A study of low level selenium determination by hydride generation atomic fluorescence spectrometry in water soluble protein and peptide fractions

Development of a method for very low level selenium determination in water soluble protein and peptide fractions, obtained after various separation procedures, is presented. A hydride generation atomic fluorescence spectrometry (HG-AFS) detection system was optimised and the influence of Cu(II), Sb(V), As(III) and HNO₃ interferences in the measurement of Se by HG-AFS was investigated. A destruction procedure using HNO₃ and H₂O₂ was also optimised and the average recovery of the digestion of a solution of selenomethioneine was 92 ± 4% (n=14). Combination of this digestion with the detection system gave reliable results. Accuracy was tested by comparison with two independent methods. A very low detection limit (DL) of 0.2 ng/g of measuring solution was achieved. The whole procedure from weighing to measuring was performed in the same Teflon tube. The addition of HNO₃ to the fractions before long term storage at -20°C was necessary to prevent adsorption on the test tubes.Selenium was measured in water soluble protein and peptide fractions obtained after extraction, and Sephadex G-75 chromatography performed on liver samples from: i) hens exposed to As₂O₃, ii) hens fed with a high fat feed and iii) the certified reference material dogfish liver (CRM DOLT-2). Because of the very low DL we were able to observe the Se distribution in chromatographic fractions of samples of organisms which were not exposed to excess amounts of Se. The presence of selenium associated with metallothioneins was observed.     

An alternative interface for CE-ICP-MS cadmium speciation in metallothioneins based on volatile species generation

An alternative CE-ICP-MS interface based on volatile species generation (VSG) is here developed, evaluated and compared to the conventional sample introduction systems via nebulisation. For this purpose, the speciation of Cd-metallothioneins (MTs) in rabbit liver is taken as a model. Cd, bound to the different MT isoforms previously separated by CE, is transformed into volatile species at the exit of the capillary and on-line detected by ICP-MS. Optimum conditions for Cd VSG have been investigated in a flow injection device, using NaBH₄ as hydrogenation reagent in a HCl medium containing cobalt and thiourea as catalysts. Sample volume injected, CE separation voltage and reagents flows have been optimised. Analytical performance characteristics of the CE-VSG-ICP-(Q)MS coupling developed were evaluated, in terms of repeatability and linearity of response, using standard rabbit liver metallothionein isoforms (MT1 and MT2). Detection limits for Cd-MTs turned out to be almost one order of magnitude better than those derived from using a conventional Babington nebuliser-based interface. Compared to a MicroMist-based interface detection limits resulted to be similar, but the observed peak height was eight times higher using the VSG interface, indicating the enhanced analyte transport efficiency derived from VSG sample introduction systems.     

Electrochemistry of and Redox‐Induced Metal Release from Metallothioneins at a Nafion‐Coated Bismuth Film Electrode

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 (E_pc=?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.     

Large volume sample stacking capillary electrophoresis for metallothioneins analysis in eel liver

A large volume sample stacking procedure (LVSS) is developed here for metallothionein (MT) determinations in rabbit liver by using capillary electrophoresis with UV detection. A 10-time improvement in concentration-based LODs, when compared to the normal stacking mode (CE-UV analysis of samples solved in water), is achieved.The methodology is successfully applied to analysis of MTs in eel liver cytosolic extracts, preceded by an easy cleaning-up pre-treatment in order to eliminate the high salt content. Analysis of cytosol obtained from a group of eels previously exposed to Cd (induced group) resulted in several isoforms of MTs with differences in absorbance signal compared to a control group.     

金属巯蛋白与人体内铜锌元素的代谢

金属巯蛋白是一类富含金属的化学结构极为特殊的内源性蛋白。它构成机体内金属元素的新陈代谢中快速应答的缓冲体系。尤其在铜、锌两种元素的吸收、转运、排泄方面发挥着重要作用。     

Zinc Transfer Kinetics of Metallothioneins and Their Fragments with Apo‐carbonic Anhydrase

The zinc transfer reactions from Zn₇‐MT‐I, Zn₇‐MT‐II, Zn₄‐α fragment (MT‐I) and Zn₄,‐α fragment (MT‐II) to apo‐carbonic anhydrase have been studied. In each reaction, no more than one zinc ion per molecule is involved in metal transfer. Zn₇‐MT‐I and Zn₇‐MT‐II donate zinc to apo‐carbonic anhydrase and de novo constitute it at a comparable efficiency, while Zn₇‐MT‐II exhibits a little faster rate. Surprisingly, Zinc is released from Zn₄‐α fragment (MT‐II) with a much faster rate than from Zn₄‐α fragment (MT‐I), whose rate is close to that of Zn₇‐MT‐I. The reason for the difference is still unknown. Introducing complex compounds into this system may give rise to an effect on the reaction. The transfer from Zn₇‐MT‐II in the presence of reduced glutathione shows little difference compare to the control, suggesting that the reduced glutathione is not involved in zinc transfer process. However, glutathione disulfide does accelerate this zinc transfer reaction remarkably, indicating that the oxidative factors contribute to zinc release from metallothioneins.     

Metal speciation of metallothionein in white sea catfish, Netuma barba, and pearl cichlid, Geophagus brasiliensis, by orthogonal liquid chromatography coupled to ICP-MS detection

Metal speciation analysis in MTs was carried out in two tropical fish species of Brazil, the freshwater fish pearl cichlid (Geophagus brasiliensis) and the marine fish white sea catfish (Netuma barba), that are presently used to monitor the effects of heavy metal pollution in aquatic ecosystems in Brazil. In order to obtain the MT fraction, liver cytosols from both fish species where subjected to size exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. That MT fraction was then separated by anion-exchange (AE)-FPLC, whose optimal chromatographic conditions were optimized for the separation of the different hepatic MT isoforms existing in both fish species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with the ICP-MS instrument. The analytical results showed that MTs of these fish species, unknown so far, exhibited unique characteristics in comparison with standard MTs and other fish liver MTs. In fact, MT isoforms of N. barba turned out to be very anionic, as indicated by their high retention in the Mono Q column and the strong ionic strength required to separate them. As for G. brasiliensis, cadmium was exclusively present in only one of the peaks of the MT isoforms showing a unique metal-binding behavior for MT in this fish species. The differences between the MTs among these species and the different association of metals in particular MT isoforms display the importance of the metal speciation analysis in these proteins prior to its use as bioindicators.     

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.     

Cyclic voltammetry study of the peptide Lys-Cys-Thr-Cys-Cys-Ala [56-61] MT i in the presence of cadmium

The electrochemical study of the peptidic fragment Lys-Cys-Thr-Cys-Cys-Ala [56–61] MT I (FT) alone and in the presence of cadmium was performed, by cyclic voltammetry using a hanging mercury drop electrode, in order to enable comparisons between the behavior of Cd, Zn metallothioneins and that of this less complex molecule, intrinsic to the metallothionein structure. The influence of the equilibration time, of the scan rate, and of the solution pH was investigated. The influence of the addition of cadmium with different Cd–FT ratios was also studied. Samples containing both Cd and FT give complex voltammograms showing different peaks. Several electrochemical systems were distinguished: the mercury electrode itself is involved in the charge transfer step in the presence of thiol groups coming from the FT structure; another system can be attributed to the oxido-reduction of cadmium either as free ion or complexed by the peptide. The adsorption phenomena play an important role in the electrochemical process. Both dissolved and adsorbed species are electroactive.     

Metal Exchange Reactions in Metallothioneins Explored by Electrochemical Methods

Metallothioneins (MTs) are widely occurring, small, cysteine‐rich proteins, important for essential metal (Zn, Cu) homeostasis and transport and for heavy metal (Cd, Hg) detoxification. In buffered solutions of mammalian MT, voltammetry and potentiometric striping analysis (PSA) can distinguish different coordination of bound metals or follow their exchange, especially that of zinc and cadmium for copper, silver, and cobalt. The examples of different electrode applications as of hanging mercury drop electrode (HMDE), of silver solid amalgam (AgSAE) electrode, and of silica gel modified carbon paste electrode (SiO₂‐CPE) are given.