SEPARATION OF METALLOTHIONEIN ISOFORMS BY FREE-SOLUTION CAPILLARY ELECTROPHORESIS IN UNCOATED AND SURFACE MODIFIED CAPILLARIES

Authors: Richards, Mark; Aagaard, Patricia

Submitted to: Federation of American Societies for Experimental Biology Conference
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/24/1994
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. MT is is comprised of a family of closely related proteins referred to as isoforms. The purpose of this study was to evaluate the effectiveness of a new technology known as Free-Solution Capillary Electrophoresis (FSCE) combined with the use of newly developed capillary coatings for separating the various forms of MT (isoforms) form the tissues of different animal species. Using this technique, it was possible to successfully isolate 4 MT isoforms from rabbit liver and horse kidney. Factors such a buffer pH and the type and concentration of the buffer were found to affect the quality of the separations of MT isoforms. In general, MT isoforms were more completely resolved by FSCE using the coated capillaries as compared to using uncoated capillaries. Together, these results offer scientists working in the area of trace element metabolism a new analytical tool (FSCE) with which to study the relationship between MT isoforms and changes in zinc and copper status.

Technical Abstract: We have recently been investigating free-solution capillary electrophoresis (FSCE) as a means to separate metallothionein (MT) isoforms. In this study, we compare the separation of MT isoforms conducted in fused-silica capillaries (50 m ID) which were either untreated (uncoated) or treated with a polyamine polymer to produce a positively charged internal capillary surface (amine-coated), or a neutral hydrophilic polyacrylamide coating (neutral-coated) which greatly reduces electroosmotic flow (EOF). FSCE in uncoated capillaries at neutral or alkaline pH resolved 2 peaks from rabbit liver MT corresponding to the MT-1 and MT-2 charge classes. At acidic pH (0.15 M sodium phosphate, pH 2.5), 3 major peaks were resolved from rabbit liver and horse kidney MT. Using the amine-coated capillary in conjunction with 0.1 M sodium phosphate buffer, pH 7.0, rabbit liver MT separated into 4 major peaks. The greater degree of MT isoform heterogeneity detected using the amine-coated capillary suggested a higher resolving capacity compared to the uncoated one. Using the neutral-coated capillary, differential resolution of rabbit liver or horse kidney MT isoforms could be achieved by performing electrophoresis toward the anode vs. toward the cathode in conjunction with an appropriate buffer. Buffer type, concentration and pH all significantly impact the resolution of MT isoforms in both uncoated and surface-modified capillaries. We conclude that FSCE conducted at alkaline pH in the uncoated capillary provides for a rapid analysis of MT-1 and MT-2 charge classes whereas FSCE in the amine- or neutral-coated capillaries provides for better resolution of some individual MT isoforms, particularly subisoforms.