Application of Capillary Zone Electrophoresis to the Separation of Metallothionein Isoforms

Abstract

Metallothioneins (MTs) from various eukaryotic species were subjected to capillary zone electrophoresis (CZE) performed under the following conditions: Capillary: untreated polyimide-clad fused silica 75 (im I.D. × 94 cm; Loading and Running Voltage: 30 kV; Electrode Buffer: 50 mM Tris-HCI pH 9.1; Sample Buffer: 10 mM Tris-HCI pH 9.1; Sample Volume: 250 μl; Detection: UV absorbance at 214 nm; Loading Method: electrokinetic migration; Loading Times: 1–6 seconds. Complete separation of MT-1 and MT-2 isoforms was achieved in less than 6 min. Using a rabbit liver Cd, Zn-MT standard (0.5 mg/ml), a linear relationship was found to exist between the voltage used to load the MT sample and the integrated peak area of the individual MT isoforms. Similarly, the integrated peak area of the separated MT isoforms was a linear function of the time used to load the sample. These findings suggest that with appropriate standardization CZE is capable of both qualitative and quantitative determinations of MT isoforms. In CZE, the concentration of MT in solution determines the limit of sensitivity. MT could be accurately detected at a concentration as low as 10 μg/ml. CZE was applied to assess thermal and proteolytic degradation of rabbit liver MT. CZE represents a rapid analytical technique for the characterization of MT isoforms that requires very little sample yet provides excellent resolution of individual MT isoforms.