Metallothioneins (MTs) belong to cysteine-rich proteins with unique higher structure. One of the most known MT's functions is metals detoxification and maintaining their homeostasis in a cell. Structure of MT with naturally occurred zinc(II) ions can be affected by concentration of metal ions as well as redox milieu inside a cell, however the exact explanation and biochemical effects of the structural changes are still missing. In this study we used capillary electrophoresis on chip coupled with fluorescence detection to determine structural changes of MT with increasing concentration of zinc(II) ions and under various redox conditions. To investigate the structural-dependent effects, reduced and/or oxidized apo-MT (MT without natural occurred metal ion) was prepared. Zinc binding into reduced and/or oxidized apo-MT was compared. MT was incubated with 0, 5, 15, 25, 50 and 100 μM ZnCl(2) for 1h in 37°C. Formation of MT aggregates with increasing zinc concentration was observed by spectrophotometry, chip capillary electrophoresis, and SDS-PAGE. We found out that reduced MT forms aggregates more readily compared to oxidized MT. Using the chip capillary electrophoresis allowed us relative quantification of MT aggregation as a decrease in the area of the signal corresponding to the monomer form of MT (Mw 15 kDa, migration time 26.5s) and its ratio to total signal (sum of all signals measured by the electrophoresis). The dependences had an exponential character with equation y=2.4×e(-0.01x), R(2)=0.945 for 15 kDa peak area and y=0.11 × e(-0.01x), R(2)=0.938 for decrease of 15 kDa peak area ratio to the total signal. Zn-MT interaction was 30% faster during the first 15 min and 50% faster during the whole experiment for reduced MT. It can be concluded that formation of MT aggregates is dependent on redox state and Zn(II) concentration.

Department of Chemistry and Biochemistry Faculty of Agronomy Mendel University in Brno Zemedelska 1 CZ 613 00 Brno Czech Republic

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