Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography

Abstract

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to isolate, characterize and quantitate liver and kidney metallothionein (MT) isoforms from a variety of animal species. The isoMTs were eluted from a radially compressed C 18 column with a neutral sodium phosphate buffer and detected by UV absorbance (214 nm). Rabbit liver and horse kidney MTs were each found to be comprised of seven distinct isoforms. Pig liver and kidney MTs each exhibited three predominant isoforms, two of which were found to be subspecies of the MT-2 isoform. Rat liver MT was characterized by a less complex isoform pattern with only two major isoforms corresponding to the MT-1 and MT-2 isoMTs. Similarly, avian liver MT exhibited a distinct isoform pattern characterized by a low degree of complexity with the MT-2 isoform much more abundant than the MT-1 species. It was possible to apply the RP-HPLC separation to the resolution of individual MT isoforms from complex mixtures such as heat-treated cytosol. A standard curve was constructed using purified turkey hen liver MT-2 which demonstrated excellent linear correlation between integrated peak area and the quantity of MT injected onto the column. Recovery of MT from RP-HPLC was estimated to exceed 90%. Liver tissue from chicks injected on consecutive days with a dose of zinc was assayed for both MT-1 and MT-2 isoforms using the RP-HPLC analysis of cytosol samples. The MT-2 isoform was found to be preferentially expressed in response to zinc induction.