The C27-steroid hydroxylating system from bovine liver mitochondria. Isolation of ferredoxin (hepatoredoxin) by affinity chromatography on cytochrome-c-sepharose
Shkumatov, V.M.; Gilevich, S.N.; Chashchin, V.L.; Akhrem, A.A.
Bioorganicheskaia Khimiia 9(9): 1231-1236
1983
ISSN/ISBN: 0132-3423 PMID: 6091698 Document Number: 215514
A technique for the isolation of highly purified hepatoredoxin involving the DE-32 cellulose chromatography of post-mitochondrial supernatant, ammonium-sulfate fractionation, Sephadex G-75 gel chromatography, 1-amino-2-hydroxypropyl-Sepharose ion-exchange chromatography and cytochrome-c-Sepharose affinity chromatography is described. The protein was purified 160-fold with a yield of 19%. The synthesis of cytochrome-c-Sepharose was carried out in a way preventing modification of the lysine-containing binding domain of the cytochrome c molecule. To achieve this, free carboxyl groups were modified with histamine to introduce imidazole residues in cytochrome c and the modified protein was immobilized on bromoacetyl-Sepharose.