Characterization of metallothionein isoforms. Comparison of capillary zone electrophoresis with reversed-phase high-performance liquid chromatography.

The purpose of this study was to compare and contrast the separation of metallothionein (MT) isoforms by reversed-phase high-performance liquid chromatography (RP-HPLC) with capillary zone electrophoresis (CZE). RP-HPLC was performed on a Vydac C8 column eluted with a linear acetonitrile gradient. CZE was performed in a 57 cm x 75 microns I.D. fused-silica tube at an operating voltage of 30 kV. Phosphate buffer (10 mM) at pH 2.5, 7.0 and 11.0 was used for both separations. CZE at pH 2.5 resolved three distinct peaks of rabbit liver MT which were incompletely resolved at pH 7.0 or 11.0. RP-HPLC at pH 2.5 gave two peaks and the resolution was not as good as with CZE at the same pH. At pH 7.0 or 11.0, RP-HPLC of rabbit liver MT gave a single predominant peak of unresolved MT-1 and MT-2. Purified rabbit liver MT-1 and MT-2 were used to verify the identity of these peaks. In contrast, MT from horse kidney demonstrated three predominant peaks which were best resolved by CZE at pH 11.0, whereas RP-HPLC resolved only two peaks at pH 11.0 and 7.0. At pH 2.5, RP-HPLC of horse kidney MT gave three peaks, though two of the peaks were incompletely separated. We conclude that pH has a considerable impact on the resolution of MT isoforms by CZE and RP-HPLC and that it is possible to exploit changes in pH to optimize the separation of isoforms for a particular species of MT. When samples of human and sheep liver MT-1, both of which exhibit microheterogeneity, were subjected to CZE, a single predominant peak was observed at each pH value. RP-HPLC of human liver MT-1 at pH 2.5 yielded two peaks that were incompletely resolved. Purified chick liver MT and rat liver MT-1 and MT-2 gave a single predominant peak at all pH values on CZE. In contrast, pig liver MT-1 and MT-2 each exhibited multiple peaks when subjected to CZE, the number of which depended on the pH used to separate the MT. In conclusion, CZE, with its orthogonal selectivity, and RP-HPLC make an excellent combination for the separation and characterization of MT isoforms. Because CZE is rapid (run times typically < 10 min) and requires little sample (< 100 nl), MT samples can readily be analyzed by CZE in conjunction with RP-HPLC or other techniques in order to maximize the information obtained about the individual isoforms.