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.
Goetghebeur
M
M
Department of Food Science and Agricultural Chemistry, McGill University, Ste Anne de Bellevue, Québec, Canada.
Kermasha
S
S
Kensley
J
J
Metche
M
M
D001234:Aspergillus niger / Q000737:chemistry*; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D003300:Copper / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006358:Hot Temperature; D007525:Isoelectric Focusing; D008668:Metallothionein / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011489:Protein Denaturation; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D014462:Ultrafiltration
D003470:Culture Media; C007121:copper thionein; D003300:Copper; D008668:Metallothionein