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A new method for the purification of cytochrome‐P450 from human liver microsomes
Author(s) -
Isa Majed,
Cumps Jean,
Fossoul Claudine,
Atassi Ghanem
Publication year - 1992
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130060510
Subject(s) - chemistry , microsome , cytochrome b5 , cytochrome p450 , chromatography , isoelectric focusing , cytochrome , yield (engineering) , biochemistry , affinity chromatography , enzyme , materials science , metallurgy
A procedure for the solubilization and purification of cytochrome‐P450 (cyt‐P450) from human liver microsomes is described. Successive treatment of microsomes with protease XXVII and 3‐(3‐cholamidopropyl)dimethylammoniopropanesulphonic acid gave a solubilized cyt‐P450 in more than 80% yield and with a three‐fold increae in specific activity. With this treatment it was possible to eliminate 80% of cytochrome‐b5 and 75% of NADPH cyt‐P450 reductase. The solubilized cyt‐P450 was filtered on a Sephacryl‐200 column and then subjected to high performance liquid chromatography with a Mono‐P column (chromatofocussing). The recovery of separated cyt‐P450 was about 50% with a specific activity of 11.5 nmol cyt‐P450/mg protein. Also with this technique it was possible to determinate the isoelectric points of cyt‐P450. These results allowed us to confirm the usefulness of our method, for the study the cyt‐P450 from surgical biopsies.