Ivana Šestáková

Recent developments in quantification methods for metallothionein

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.

Differential pulse voltammetric study of the complexation of Cd(II) by the phytochelatin (γ-GluCys)2Gly assisted by multivariate curve resolution

Abstract A multivariate curve resolution method by alternating least-squares (MCR-ALS) is applied to differential pulse voltammograms measured on the Cd(II)+(γ-GluCys) 2 Gly system as a model of metal–phytochelatin interactions at concentrations of both components in the range 10 −7 –10 −5 mol l −1 . The course of complexation is different when peptide is titrated with metal from that when metal is titrated with peptide. The combined analysis of both matrices from titrations of peptide with metal and of metal with peptide allowed the resolution of the system. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggested the presence of four different types of bound Cd(II) and made possible the formulation of a complexation model.

Voltammetric methods in metallothionein research.

The application of voltammetric methods using different rates of polarisation on HMDE reveal inert or labile behaviour of Cd- or Zn- complexes in the presence of excessive cadmium or zinc ions in solution. This phenomenon was demonstrated first on the simplest phytochelatin – complex of peptide (γ-Glu-Cys)2 Gly with cadmium, later on rabbit liver metallothioneins – Cd7 MT in the presence of cadmium and Cd5 Zn2 MT in the presence of zinc. Voltammetric methods can distinguish between labile and inert complexes present simultaneously and therefore could elucidate their role in reactions of metal ion transfer. Another method using different rates of polarisation – elimination voltammetry with linear scan – proved that S-tetracoordinated complexes of Cd(II) or Zn(II) in the above-mentioned metallothioneins on HMDE are reduced in the adsorbed state. This implies the possibility of increasing the sensitivity of identification or determination of the above complexes. On carbon composite electrode, similar behaviour of Cd-complexes as on HMDE was observed using differential pulse voltammetry.

Adsorption of Cd,Zn-metallothionein on covered Hg electrodes and its voltammetric determination

Abstract Compact triphenylphosphine oxide or tripiperidinophosphine oxide films formed at Hg surfaces are shown to increase Cd, Zn-metal-lothionenin (MT) adsorptivity at the polarized electrode/solution interface. Interactions of the polar P=O group of these adsorbates with the hydrophilic centers located on the metalloprotein exterior are suggested as being responsible for this phenomenon. The resulting cathodic current ( i c ) due to the reduction of coordinated Cd(II) in Cd,Zn-MT increases more than seven times in the presence of 1.0 × 10 −3 M triphenylphosphine oxide. The i c response at about −0.72 V vs. Ag/AgCl then varies linearly with the bulk concentration of Cd,Zn-MT from 2 × 10 −9 to 5 × 10 −5 mol dm −3 . Detection limit for the rabbit liver Cd,Zn-MT is found to be 8.9 × 10 −10 mol dm −3 using the differential pulse voltammetric technique on a hanging mercury drop electrode. This novel method for the detection of the thiolate-chelated Cd(II) complexes allows the filtering out of cathodic currents due to electroreduction of the free-uncoordinated Cd 2+ ions which might interfere with the MT determination in some biological samples. Advantages of the present method compared to other electroanalytical methods for MT determination are discussed.

The use of differential pulse anodic stripping voltammetry and diffusive gradient in thin films for heavy metals speciation in soil solution

In the soil solutions obtained in situ with suction cups from soils (Cambisol and Fluvisol) of pot experiment with Salix smithiana Smith, Lolium perenne L. and Thlaspi caerulescens J. & C. Presl heavy metals species (Cd, Pb and Cu) were assayed by differential pulse anodic stripping voltammetry and diffusive gradient in thin films. Prediction of accumulation performed best at free metal ion concentrations in unchanged pH (in 10−3 mol L−1 NaClO4 base electrolyte). The speciation provided by differential pulse anodic stripping voltammetry according to pH can provide a detailed description of the soil solution matrix. The concentration of free metals in unchanged pH represents a small part of the total content and varied from 0.04 to 0.75% with two exceptions found for accumulating plants (the content of Cd2+ in the soil solution from T. caerulescens was about 6% and the content of Cu2+ in the soil solution from S. smithiana was about 30%). The available concentration as determined by diffusive gradient in thin films was not in correlation with the heavy metals concentration in plant biomass.

A new approach to study cadmium complexes with oxalic acid in soil solution

This study presents a new analytical approach for the determination of heavy metals complexed to low-molecular-weight-organic acids in soil solutions, which combines the sensitivity of differential pulse anodic stripping voltammetry (DPASV) with the molecular insight gained by electrospray ionization mass spectrometry (ESI-MS). The combination of these analytical methods allows the investigation of such complexes in complex matrixes. On the voltammograms of the soil solutions, in addition to the expected complexes of oxalic acid with cadmium and lead, respectively, also peaks belonging to mixed complexes of cadmium, lead, and oxalic acid (OAH(2)) were observed. In order to verify the possible formation of complexes with OAH(2), aqueous solutions of OAH(2) with traces of Cd(II) were investigated as model systems. Signals corresponding to several distinct molecular complexes between cadmium and oxalic acid were detected in the model solutions using negative-ion ESI-MS, which follow the general formula [Cd(n)(X,Y)((2n+1))](-), where n is the number of cadmium atoms, X=Cl(-), and Y=OAH(-). Some of these complexes were also identified in the ESI mass spectra taken from the soil solutions.

Characterization of cadmium ion transport across model and real biomembranes and indication of induced damage of plant tissues

The present study is focused on characterization and elucidation of transport processes of the environmentally important element cadmium across model and real membranes. Basic steps of these processes were clarified on model synthetic membranes, which were characterized by atomic force microscopy and by electrochemical impedance spectroscopy. To gain the cytoplasmic membranes containing specific transport systems, the technique for isolation of protoplasts from barley cells was optimized (cell wall-degrading enzyme, physiological state of plant leaves, type of osmotic stabilizers, and the composition of reaction solution). The obtained real parts of membranes were mixed with lecithin membranes and were characterized by electrochemical impedance spectroscopy, and the transported species were determined by voltammetric methods. Further, influence of cadmium on damage of plant tissues was investigated. Young barley plants were subjected to different doses of cadmium chloride and its presence affected the tissue damage, which could be characterized via conductivity of the electrolyte leakage.Graphical abstract

Voltammetric detection of phytochelatin transported across unmodified and protoplast modified model phospholipid membranes

Possibility of phytochelatin PC2 transport was examined using developed electrochemical cell with two compartments separated by a phospholipid membrane created in pores of polycarbonate support. At first cell penetrating peptides (transportan 10 and mastoparan X) were used to allow the transfer of phytochelatin PC2. The results demonstrated that transportan 10 is several times more efficient than mastoparan X for transferring of phytochelatin across model membrane. Voltammetry with hanging mercury drop electrode or with solid amalgam electrodes was applied for detection of transported species. For determination of PC2, hanging mercury drop electrode and mercury meniscus-modified silver solid amalgam electrode were suitable. Modified model phospholipid membrane was prepared with parts of protoplasts from barley roots or leaves, and transport of phytochelatin PC2 has been confirmed. Electrochemical impedance spectroscopy was utilized to monitor formation, stability, and qualitatively the transporting process across the supported phospholipid membranes.Graphical abstract

Voltammetric methods in isolation and identification of plant metallothioneins from alga Chlorella

In response to the environmental pollution by metals such as Cd, Cu, Pb, Ni, and As, special thiol-rich peptides are synthesized in plants and certain fungi. Their general structure is (γ-Glu-Cys)n-R, where R is an amino acid residue (Gly, Ala, Ser, Glu). These peptides are known as plant metallothioneins, class III metallothioneins or γ-EC peptides [1]. Cd and Cu are the metals most frequently bound to plant metallothioneins from different sources and are well characterized by UV spectroscopy due to special absorption of the Cd-S and Cu-S bonds. Another possibility is offered by voltammetric methods which can distinguish between free metal ion and metal ion bound in complexes of different stability. For Cd complexes of γ-EC peptides structural models have been proposed [2] in which each cadmium ion is coordinated by four sulfur atoms. No voltammetric studies have been performed with γ-EC peptides so far. With animal metallothioneins, where tetracoordinated cadmium is present in molecules consisting of 60-62 amino acids, several polarographic and voltammetric studies have been performed. In our first study [3], we worked with rabbit liver metallothionein-2, prepared from rabbits exposed to cadmium salt, and with Cd-MT and apo-MT prepared from rabbit. We compared the voltammetric behavior on several types of carbon electrodes with that on the hanging mercury drop electrode (HMDE); active role of mercury electrode, confirmed also in other papers, can be best demonstrated with cyclic voltammetry (Fig. 1). An extreme reactivity of thiols with mercury is well known and detailed studies on reactions of cystine [4] and cysteine [5] have been performed. Also, it has been demonstrated, that under certain conditions, compact triphenyphosphine oxide films formed at the Hg/solution interface, can block thiol or disulfide adsorption on Hg electrodes [4].