Purification and characterization of copper-metallothioneins from Aspergillus niger

Copper metallothioneins were extracted from Aspergillus niger after an induction period of five hours in a copper-containing suspension medium. The crude extract (FI) was obtained by cell homogenization and the partially purified extract (FII) was obtained by heat treatment at 60°C for 10 min. The partially purified extract (FII) was further purified by successive chromatographies on size-exclusion (Biogel P-30) and ion-exchange (TSK-HW 65-DEAE) chromatography columns. All protein fractions were analyzed for their protein and induced copper contents. The Cu/protein ratio increased steadily throughout the purification process; the most purified fraction (FIV) showed a 24-fold increase, with a recovery of 13%. The molecular weight of the Cu-metallothionein fraction FIV was 11,000 and the ratio of moles of copper per mole of protein was 6.6.