SEPARATION OF 3 MOUSE METALLOTHIONEIN ISOFORMS BY FREE-SOLUTION CAPILLARY ELECTROPHORESIS

We have used free-solution capillary electrophoresis (FSCE) to separat e three distinct mouse metallothionein (MT) isoforms, MT-1, MT-2 and M T-3. FSCE was conducted in an uncoated fused-silica capillary (57 cmXS O mu m I.D., 50 cm to detector) using 50 mM sodium phosphate buffer ad justed to pH 7.0 or 2.0. At neutral pH, each of the three isoform peak s were well resolved from a mixture with the order of migration (MT-1> MT-2>MT-3) related to the net negative charge on the protein. At acidi c pH, the migration order was reversed with MT-3 migrating fastest, su ggesting MT-3 had a higher net positive charge than MT-2 or MT-1. UV a bsorbance spectra (190-300 nm) confirmed the presence of Zn in MT-1 an d MT-2. MT-3, which was saturated with Cd to stabilize the protein, ga ve a spectrum characteristic of the Cd-S charge transfer (shoulder at ca. 250 nm). At pH 2.0, the absorbance spectra for all three mouse MTs were characteristic of the metal-free form of the protein (apothionei n). Thus, FSCE conducted at neutral pH separates MT isoforms with thei r metals intact, whereas at pH 2.0, both the Zn and the Cd dissociate from the protein during the run.