Characterization of Mammalian Metallothionein Isoforms by Capillary Zone Electrophoresis With Diode Array Detection Using Tris-Borate Buffer

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Abstract
Four mammalian metallothioneins (MTs), rabbit liver MT (RL MT), rabbit liver MT-1 (RL MT-1), and rabbit liver MT-2 (RL MT-2) and horse kidney MT (HK MT), were subjected to capillary zone electrophoresis (CZE) with on-line diode array detection (DAD) by using tris-borate buffer at neutral or nearly neutral conditions thus, giving information about the putative isoforms in conditions also found in tissues. The MTs were found to exhibit a different polymorphism. Rabbit liver MT exhibited 3 main peaks and horse kidney 5 main peaks. Isoform abundance between batches was found to vary noticeably. Differences in electrophoretic behaviour were observed. As expected, the 2-charged main peak of RL MT-2 showed highest electrophoretic mobility. Main peak of RL MT-1 and two of rabbit liver MT had lower electrophoretic mobilities corresponding to single charge. Two later migrating horse kidney MT peaks had nearly similar electrophoretic behaviour as the single charged rabbit liver MT peaks. Interestingly, horse kidney MT exhibited also peaks with lower electrophoteric mobilities and different behaviour than the other peaks observed. The ultra violet (UV) spectra obtained by using diode array detection (DAD) showed clear difference in the structure of these peaks compared to other peaks observed. These peaks showed no, or nearly no, absorbance at wavelengths 225 and 250 nm that corresponds to cation-thiol bondage, Zn-thiol, and Cd-thiol, respectively. We can, therefore, assume that horse kidney MT sample contains components that are in apothionein form are only partially saturated with metals. The use of diode array detection appears to be a good tool for a quick identification of apothioneins and partially metal-saturated metallothioneins.

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