Separation of metallothionein isoforms by capillary zone electrophoresis

The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 microns I.D. x ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 mM Tris-HCl buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 mM Tris-HCl, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 micrograms/ml-1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1-10 micrograms MT ml-1. The successful use of two solvent extraction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.