Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis.
We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60-80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.
Richards
M P
MP
USDA, Agricultural Research Service, Growth Biology Laboratory, Beltsville, MD 20705-2350, USA.
Beattie
J H
JH
D000818:Animals; D002645:Chickens; D019075:Electrophoresis, Capillary / Q000379:methods*; D007536:Isomerism; D008668:Metallothionein / Q000032:analysis*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D008668:Metallothionein