Determination and purification of metallothioneins by high performance liquid chromatography

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Abstract
A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein‐I (MT‐I) and metallothionein‐II (MT‐II) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT‐I or MT‐II was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT‐I and MT‐II were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd‐injected samples and 60Co‐irradiated samples. A detection limit of 5 μg/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.