Author
 |
Richards, Mark |
|
Submitted to: Capillary Electrophoresis Conference Proceedings
Publication Type: Abstract Only Publication Acceptance Date: 10/25/1994 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Metallothioneins (MTs) are a group of low molecular weight, heavy-metal-binding proteins that function in intracellular metal metabolism. The objective of this work is to develop and optimize quantitative capillary electrophoresis (CE)-based techniques for the analysis of MT isoforms. At alkaline pH, CE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH (2-2.5), additional resolution of MT isoforms is achieved, perhaps due to the fact that metals dissociate from MTs and the apo-proteins (thioneins) are subsequently separated. Capillary diameters (I.D.) of 75 or 50 um permit a lower limit of detection (LOD) of about 1-5 ug MT/ml. Although 20 um I.D. capillaries exhibit larger LODs, they allow for the use of higher ionic strength (0.5 M) phosphate buffers that result in higher peak efficiencies and increased resolution for MT isoforms. Interior capillary surface coatings such a linear polyacrylamide and polyamine polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow and potential protein-wall interactions. Enhanced MT isoform resolution can also be achieved by micellar electrokinetic capillary chromatography (MECC) using 100 mM borate buffer pH 8.4 containing 75 mM SDS. This technique is particularly useful in the analysis of crude tissue extracts containing MT. Sample preparation prior to CE has been investigated. Deproteinization with acetonitrile (60-80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by CE or MECC. In conclusion, the analysis of MT isoforms by CE has been achieved through a combination of different capillaries, buffers and sample preparation techniques. |
