A new method for the purification of cytochrome-P450 from human liver microsomes.
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.
Isa
M
M
Laboratoire de Pharmacologie Cellulaire, Université Libre de Bruxelles, Belgium.
Cumps
J
J
Fossoul
C
C
Atassi
G
G
D001706:Biopsy; D002793:Cholic Acids; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015786:Cytochromes b5 / Q000302:isolation & purification; D003902:Detergents; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D010450:Endopeptidases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate