QUANTIFICATION OF BASAL AMOUNTS OF ISOMETALLOTHIONEINS IN CULTURED-CELLS BY REVERSED-PHASE HPLC

A reversed phase HPLC method was developed to separate and quantify ba sal amounts of the major isometallothionein, i.e. MT-2, in cell extrac ts of human liver cells. To separate MT-2 from interfering proteins an ultrafiltration step was included in the preparation of the sample. C alibration was performed with varying amounts of pure human MT-2 quant ified 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 ob tained. Linear correlation between the amount applied and the integrat ed area resulted when MT-2 was diluted with cytosol equivalent to 5.5 x 10(6) cells. The detection limit under these conditions was 5 pmol, sufficient to monitor basal metallothionein concentrations and to foll ow changes in the accretion of these proteins under physiological cond itions.