ANALYSIS OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ELECTROPHORESIS - OPTIMIZATION OF PROTEIN SEPARATION CONDITIONS USING MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY

Capillary electrophoresis (CE) techniques have been successfully appli ed to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer compos ition and capillary wall surface modifications can have considerable i nfluence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrati ons and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electr ic field is the basis of the technique known as micellar electrokineti c capillary chromatography (MECC). In the present work, we have used s heep and rabbit MT to optimise MECC conditions for analysis of MT isof orms. Capillaries of 57 cm gave much better separations than shorter c olumns although analysis times were increased to about 12 min. Changin g the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diamete r capillaries we conclude that 25 mu m I.D. columns give better separa tions than 50 or 75 mu m I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C-1 or C-18 hydrophobic material were not found t o be useful in improving MT separation or resolution although further evaluation of these columns is in progress. Analysis of sheep liver ex tracts using optimised conditions showed the expression of at least 4 MT isoforms in response to Zn injection and 3 of these forms were evid ent in extracts from untreated sheep. We therefore conclude that MECC is a suitable method for MT isoform analysis.