Characterization of isoprotein patterns in tissue extracts and isolated samples of metallothioneins by reverse-phase high-pressure liquid chromatography

Abstract

Reverse-phase high-pressure (‘performance’) liquid chromatography was used to characterize the isometallothioneins in preparations isolated from tissues of a variety of animals by ‘conventional’ chromatographic methods. The resolution was such that isoproteins differing by a single serine leads to leucine difference in 61 residues could be easily separated. Yields from the reverse-phase support were typically 60-70% for the isoproteins. Comparisons of isometallothionein patterns after Cd2+-induction in rabbits indicated that total metallothionein concentrations were about 4-fold higher in liver than kidney extracts from the same animal. In the extracts a minimum of four and six isometallothionein peaks were detected in kidney and liver respectively. Under acidic conditions, where the metals are removed from the protein, the chromatographic properties, i.e. hydrophobicities, of the isoproteins from kidney were identical with those of four of those found in liver. Although the same peaks appeared in tissue extracts from individual animals, concentration differences were apparent. Remarkably, no differences were observed between the isoprotein patterns of liver or kidney as a consequence of either Cd2+- or Zn2+-induction. Chromatography of the metal-containing forms at neutral pH in Tris buffer indicated that the relative ratios of the complexed metal ions in the isoproteins were found to be effectively identical, not only before and after chromatography, but also within the separated forms from a single tissue source.