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Title: A COMPARISON OF DIFFERENT TECHNIQUES FOR THE ANALYSIS OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ELECTROPHORESIS

Author
item Richards, Mark
item BEATTIE JOHN H - THE ROWETT RESEARCH INST.

Submitted to: Capillary Electrophoresis Conference Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/1994
Publication Date: N/A
Citation: N/A

Interpretive Summary: Trace elements such as zinc and copper are required to support growth and development in all animal species, including humans. There has been a great deal of interest in the scientific community in developing indicators of zinc and copper status. One such status indicator is a zinc- and copper-binding protein called metallothionein (MT). Although MT has been actively studied for over thirty years, there continues to be a need for new rapid and more sensitive methods to assay levels of MT in physiological fluids such as blood as well as in tissues such as liver and kidney. The purpose of this study was to compare and contrast different methodologies for isolating and quantifying MT by a new technique called capillary electrophoresis (CE). We evaluated specific modifications to the capillary surface and to the buffer used to perform the analysis. In addition, different methods of sample preparation were studied to optimize the detection sensitivity for MT in liver tissue extracts. The results from this study help to advance the state of CE technology for its specific application to MT analysis and offer new information useful in formulating an assessment of trace element status based on analyzed MT levels.

Technical Abstract: We have been investigating 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. The objective of this work is to develop and optimize quantitative CE-based techniques for the analysis of MT isoforms. 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, perhaps due to the fact that metals dissociate from MTs and the metal-free proteins are subsequently separated. Use of capillaries with internal 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 give higher LODs, they allow for the use of higher ionic strength (0.5 M) phosphate buffers that can result in higher 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. 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 FSCE or MECC. We conclude that the analysis of MT isoforms by CE can be achieved through a combination of different capillaries, buffers and sample preparation techniques.