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-exchang e (AE) high-performance liquid chromatography (HPLC) techniques were e mployed in combination with on-line radioactivity detection, in a stud y on the kinetic behaviour of Zn-65-, Cu-64- and [S-35]cystein-labelle d metallothionein (MT) in rat hepatoma tissue culture (HTC) cells. MSE -HPLC of [S-35]cysteine-labelled HTC cell cytosol resulted in co-eluti ng MT-I and MT-II isoforms (t(R) 19.80 min; V(e)/V(o) 1.85). AE-HPLC o f 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 ac id composition, apparent molecular mass and cysteine content of HTC MT -I and MT-II isoforms showed the characteristics of class I metallothi oneins. The rate of dissociation of Zn2+ from Zn-MT could be determine d from the losses of Zn-65 from MT during a single AE-HPLC run, showin g 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 th e appearance of reduced glutathione as an intracellular copper-transfe r compound. Application of compartmental analysis in [S-35]-cysteine a ccumulation experiments permitted the determination of the actual rate of MT degradation, when 200 muM of Zn were applied, the MT degradatio n 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.