Capillary electrophoresis of metallothionein

Metallothionein (MT) was first isolated in 1957 from the horse kidney by Margoshes and Vallee [1], subsequently MT presence was demonstrated in other animals, in higher plants, eukaryotic microorganisms, and in some prokaryotes [2]. Mammalian MT belongs to a group of low molecular weight proteins (6000-7000 Da) with 30% content of cysteine. A characteristic feature of all MTs is a tripeptide sequence Cys-Xaa-Cys, where Xaa is an aminoacid other than cysteine [3]. The sulfhydryl groups of cysteine form with metals closely packed spatial structure in which metals are within the molecule [2,4]. It serves as the reservoir of metals for the body (mainly Zn and Cu) which are the part of many enzymes and proteins involved in the removal of DNA damage, replication or transcription. MT protects the cells against the toxicity of heavy metals (Cd, Pb, Hg) by binding them. MT is composed of two domains: α and β linked by lysine dimer. Domain α (C-terminal) is more stable and is able to bind four atoms of Zn or Cd, or five or six atoms of Cu. Beta domain (N terminal) is more reactive, it can bind tri- Zn or Cd, or six atoms of Cu [4]. Slight differences in amino acid composition, hydrophobicity and isoelectric point allowed to separate the four major isoforms: MT-1, MT-2, MT-3 (also known as, growth inhibitory factor GIF), and MT-4. Despite the mutual similarity of MT-1 and MT-2, their roles and presence in tissues differ significantly [5]. While MT-2 isoform in humans is encoded by a single gene MT-2A, MT-1 consists of multiple subtypes encoded by a gene set of MT-1 (MT-1A, 1B-MT, MT-1E, MT-1F, MT-1G, 1H and MT-MT-1X), which determine the micro-heterogeneity of the protein (Figure 1).

< p class="juxe"> MT-1 and MT-2 genes expression have been demonstrated in many tissues: in the parenchymal cells of the kidney, liver, lung, intestines, pancreas [7], in cells of the sweat glands of the skin, in germ cells, and in epithelial cells of the thymus [8]. Izofom MT-3 is a protein mainly present in brain tissue [9]. The expression of MT-4 is limited to squamous skin and upper gastrointestinal [10]. Sites of presence of particular isoforms of human MT are listed in Table 1 [11]. MT cysteine residues also play a critical role in the removal of free radicals. MT has antitumor activity as it neutralizes toxic electrophiles, reactive oxygen and nitrogen species. MT is also capable of inhibiting apoptosis and developing resistance to radiotherapy and chemotherapy. Higher expression of this protein has been observed in many tumor tissues: lung, kidney, pancreas, bladder, urinary tract. Lower concentration levels have been observed in gastrointestinal tumors tract, including liver cancer or colon cancer.

< p class="juxe"> To identify MT, sensitive and selective analysis techniques are applied. Low molecular weight of protein, and the heterogeneity of the biological material to be analyzed (serum, erythrocyte lysate, urine, different tissues), also causes a variety of methods used for its quantitative determination.

< p class="juxe"> Electrochemical methods such as differential pulse and cathodic stripping voltammetry, saturation analysis methods based on Cd, Ag and Hg, spectrophotometric methods as well as chromatographic and electrophoretic techniques are used [12]. Among immunological techniques used in MT analyses are: enzyme-linked immunosorbent assay, immunofluorescence assay and radioimmunoassay. These methods are highly sensitive and can detect even small amounts of MT in tested material [13,14]. One of the most widely used techniques in the analysis of proteins including MT is two-dimensional polyacrylamide gel electrophoresis or capillary electrophoresis (CE) often coupled with mass spectrometry (MS). CE allows analysis of both MT as MT in complexes with metals. As the determination of various isoforms is concerned, there must be very sophisticated detection system connected with separation one. Today, medicine has a lot of interest in research on this protein, and thanks to modern analytical methods with high sensitivity and specificity, the determination of MT concentrations in the biological material is possible, wherein its amount in relation to other proteins is small.

Figure 1: Comparison of the amino acid sequences of MT 1/2 human isoforms using the ClustalX program 2.0.11 [6].

Table 1: Places of expression of particular isoforms of human MT [Based on 11].

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