A new method for the purification of cytochrome-P450 from human liver microsomes. |
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Authors: | M Isa J Cumps C Fossoul G Atassi |
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Affiliation: | Laboratoire de Pharmacologie Cellulaire, Université Libre de Bruxelles, Belgium. |
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Abstract: | 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 increase 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 determine 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. |
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