Isolation and quantitation of cadmium-, zinc- and copper-metallothioneins by high-performance liquid chromatography-atomic absorption spectrometry

Abstract

Metallothioneins (MTs) were separated and quantitated by reversed-phase high-performance liquid chromatography (RP-HPLC), in direct combination with atomic absorption spectrometry (AAS) for quantitation of the metal contents in MTs. MTs were eluted from an RP-8 column with a gradient of Tris buffer pH 7.0 and methanol, and were detected by UV absorbance (220 nm). Commercially available purified MTs from horse kidney and rabbit liver were analyzed for purity and metal composition. One lot of horse kidney yielded only 50% of the estimated value. In some cases, the certified metal content differed considerably from the values found. The method was also tested with rat liver and fetal bovine liver. The metal contents found in MTs by use of graphite-furnace atomic absorption spectrometry corresponded well with the values found by RP-HPLC-AAS. Molar ratios of cadmium, zinc and copper were calculated in MT-1, MT-2 and total MT.