Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography.
NADPH:cytochrome c (cytochrome P-450) reductase (Fp) from hamster liver microsomes has been purified to near homogeneity using a simple and rapid method. Microsomes were treated with the detergent Chaps (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid) in combination with 0.07% protamine sulfate and then centrifuged to pellet insoluble material. While over 60% of the total microsomal protein was solubilized, all Fp activity remained in the pellet. Fp was extracted from the Chaps-insoluble material using a combination of the detergents sodium cholate and Lubrol PX. This treatment resulted in a fivefold increase in Fp specific activity and allowed direct processing of the enriched Fp fraction by 2',5'-ADP agarose affinity chromatography. The purified Fp had a total flavin content of 23 nmol/mg protein (flavin adenine dinucleotide:flavin mononucleotide ratio = 1:1), a specific activity of 26,000 units/mg protein at 22 degrees C using cytochrome c as electron acceptor, and migrated as a single band on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis with a relative molecular weight of 76,000. The purity, specific activity, and yield were nearly identical to results obtained when the flavoprotein was purified by conventional methods. This procedure eliminates the need for anion-exchange chromatography and allows for the rapid purification of large amounts of Fp suitable for use in studies concerning cytochrome P-450-mediated drug metabolism. Importantly, this method is equally effective when used to purify Fp from rat liver microsomes.
D000818:Animals; D002793:Cholic Acids; D002846:Chromatography, Affinity; D006224:Cricetinae; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification*
D002793:Cholic Acids; D003902:Detergents; D009251:NADPH-Ferrihemoprotein Reductase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate