Determination of zinc-65, copper-64 and sulphur-35 labelled rat hepatoma tissue culture metallothioneins by high-performance liquid chromatography with on-line radioactivity detection

Abstract

Molecular size exclusion (MSE), reversed-phase (RP), and anion-exchange (AE) high-performance liquid chromatography (HPLC) techniques were employed in combination with on-line radioactivity detection, in a study on the kinetic behaviour of 65Zn-, 64Cu- and [ 35S]cystein-labelled metallothionein (MT) in rat hepatoma tissue culture (HTC) cells. MSE-HPLC of [ 35S]cysteine-labelled HTC cell cytosol resulted in co-eluting MT-I and MT-II isoforms ( t R 19.80 min; V e/ V o: 1.85). AE-HPLC of 65Zn-treated HTC cell cytosol yielded separated 65Zn MT-I ( t R 11.5 min; I = 64 m M) and 65Zn MT-II ( t R 14.5 min; I = 104 m M). RP-HPLC of 64Cu-treated HTC cytosol resulted in separated 64Cu MT-I ( t R 26.4 min) and 64Cu MT-II ( t R 23.4 min). Determination of the amino acid composition, apparent molecular mass and cysteine content of HTC MT-I and MT-II isoforms showed the characteristics of class I metallothioneins. The rate of dissociation of Zn 2+ from Zn-MT could be determined from the losses of 65Zn from MT during a single AE-HPLC run, showing a Zn-MT dissociation half-life of 0.66 h. RP-HPLC showed a delay in incorporation of newly accumulated 64Cu into MT, possibly owing to the appearance of reduced glutathione as an intracellular copper-transfer compound. Application of compartmental analysis in [ 35S]-cysteine accumulation experiments permitted the determination of the actual rate of MT degradation; when 200 μ M of Zn were applied, the MT degradation half-life was 2.0 ± 0.8 h. These results indicate the potential of combined HPLC techniques and application of radionuclides in studies on the synthesis and degradation of MT and metal-MT complexes.