Purification and characterization of copper-metallothionein from Aspergillus niger by affinity chromatography

Biomass of Aspergillus niger was obtained from a microbial culture and a copper (Cu) induction was performed after 72 h of fermentation. The crude induced metallothionein extract was obtained by cell disruption and partly purified by a heat treatment and ultrafiltration. The purification of metallothionein by affinity chromatography resulted in three major fractions: FIVa, FIVb and FIVc3. Cu analysis demonstrated that only fraction FIVc contained the Cu-metallothionein. Spectrophotometric analyses of FIVc demonstrated the presence of a peak at 259 nm and a ratio of 78 mol of Cu per mol of protein. Electrophoretic analyses of FIVc, performed under denaturing conditions, showed the presence of one band with molecular mass of 21 kDa; however, two isoforms were observed under native conditions with molecular masses of 9.5 and 10.5 kDa and isoelectric points of 6.2 and 6.5, suggesting a recombination process due to denaturation.