Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography.
Capillary electrophoresis (CE) techniques have been successfully applied 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 composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing 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 diameter capillaries we conclude that 25 microns I.D. columns give better separations than 50 or 75 microns I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C1 or C18 hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress.(ABSTRACT TRUNCATED AT 250 WORDS)
Beattie
J H
JH
Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, Scotland.
Richards
M P
MP
D000818:Animals; D002197:Capillary Action; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004573:Electrolytes; D004586:Electrophoresis / Q000379:methods*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry / Q000302:isolation & purification*; D008823:Micelles; D011817:Rabbits; D012756:Sheep; D012967:Sodium Dodecyl Sulfate
D004573:Electrolytes; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D008668:Metallothionein