DETERMINATION OF ZN-65, COPPER-64 AND S-35 LABELED RAT HEPATOMA TISSUE-CULTURE METALLOTHIONEINS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ONLINE RADIOACTIVITY DETECTION

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 Zn-65-, Cu-64- and [S-35]cystein-labelled metallothionein (MT) in rat hepatoma tissue culture (HTC) cells. MSE-HPLC of [S-35]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 Zn-65-treated HTC cell cytosol yielded separated Zn-65 MT-I (t(R) 11.5 min; I = 64 mM and Zn-65 MT-II (t(R) 14.5 min; I = 104 mM. RP-HPLC of Cu-64-treated HTC cytosol resulted in separated Cu-64 MT-I (t(R) 26.4 min) and Cu-64 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 Zn2+ from Zn-MT could be determined from the losses of Zn-65 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 CU-65 into MT, possibly owing to the appearance of reduced glutathione as an intracellular copper-transfer compound. Application of compartmental analysis in [S-35]-cysteine accumulation experiments permitted the determination of the actual rate of MT degradation, when 200 muM 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.