Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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Advances in Clinical and Experimental Medicine

2018, vol. 27, nr 11, November, p. 1601–1608

doi: 10.17219/acem/98916

Publication type: original article

Language: English

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The application of capillary electrophoresis, mass spectrometry and Brdicka reaction in human and rabbit metallothioneins analysis

Marta Kepinska1,A,B,C,D,E,F, Sona Krizkova2,3,B,F, Ewelina Guszpit1,B,F, Miguel A. Merlos Rodrigo2,3,B,F, Halina Milnerowicz1,E,F

1 Department of Biomedical and Environmental Analysis, Faculty of Pharmacy with Division of Laboratory Medicine, Wroclaw Medical University, Poland

2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Czech Republic

3 Central European Institute of Technology, Brno University of Technology, Czech Republic

Abstract

Background. Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells.
Objectives. The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared.
Material and Methods. Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization.
Results. In chip CE analysis, changes in the range of 5–8 kDa corresponding to the MT monomer, as well as some peaks of 13–14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT.
Conclusion. The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of –sulfhydryl (−SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.

Key words

metallothionein, mass spectrometry, capillary electrophoresis, Brdicka reaction

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