APPLICATION OF A POLYAMINE-COATED CAPILLARY TO THE SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS

In this study a fused-silica capillary treated internally with a polyamine coating which reverses electroosmotic flow in the direction of the anode was evaluated for its ability to resolve metallothionein (MT) isoforms. Analysis of different MTs purified from liver and kidney tissue revealed the following numbers of putative isoform peaks resolved: rabbit (3-6); horse (3-5); rat (2-3), chicken (1); human MT-1 (5-6); sheep (4-5) and pig (4-5). The greater degree of MT isoform heterogeneity detected in this study using the polyamine-coated capillary suggested a higher resolving capacity for capillary zone electrophoresis conducted with this capillary compared to an uncoated one. Using the single isoform of chicken MT (cMT) as a reference standard, relative standard deviations of 2.53, 1.85 and 2.21% for peak migration time, area and height, respectively, were observed for eight consecutive runs. A standard curve for cMT established linearity (r(2) = 0.99) for integrated peak area over three log units of cMT concentration with a lower limit of detection estimated to be less than or equal to 5 mu g/ml. Acetonitrile extracts of chick liver tissue homogenates were successfully analyzed for the presence of MT isoforms from both control and zinc-injected animals. Based on our initial evaluation, capillary zone electrophoresis using the polyamine-coated capillary appears to be a very useful analytical method for the separation and quantification of individual MT isoforms.