Quantification of Basal Amounts of Isometallothioneins in Cultured Cells by Reversed-Phase HPLC

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Abstract
A reversed phase HPLC method was developed to separate and quantify basal amounts of the major isometallothionein, i. e. MT-2, in cell extracts of human liver cells. To separate MT-2 from interfering proteins an ultrafiltration step was included in the preparation of the sample. Calibration was performed with varying amounts of pure human MT-2 quantified by amino acid analysis. Using MT-2 diluted with 25 mM Tris/HCl a non-linear calibration curve with a detection limit of 70 pmol was obtained. Linear correlation between the amount applied and the integrated area resulted when MT-2 was diluted with cytosol equivalent to 5.5 × 106 cells. The detection limit under these conditions was 5 pmol, sufficient to monitor basal metallothionein concentrations and to follow changes in the accretion of these proteins under physiological conditions.