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A two‐step purification of cytochrome P‐450 from adult pig testis by pregnenolone affinity column chromatography
Author(s) -
Kuwada Masahiro
Publication year - 1999
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/(sici)1099-0801(199908)13:5<344::aid-bmc887>3.0.co;2-7
Subject(s) - chemistry , chromatography , cytochrome , isoelectric focusing , isoelectric point , sodium dodecyl sulfate , pregnenolone , affinity chromatography , polyacrylamide gel electrophoresis , column chromatography , cytochrome c , gel electrophoresis , yield (engineering) , biochemistry , steroid , mitochondrion , enzyme , hormone , materials science , metallurgy
Adult testicular cytochrome P‐450 was purified by a two‐step procedure utilizing hydroxylapatite and pregnenolone affinity column chromatography. The cytochrome P‐450 was determined to have an isoelectric point of 6.43 on analytical isoelectric focusing. The purified cytochrome P‐450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P‐450. A purification of 755× was achieved with a yield of 3.09%. Copyright © 1999 John Wiley & Sons, Ltd.Abbreviations used : EAH‐Sepharose 4B, 1,6‐diaminohexyl‐sepharose 4B;Emulged 913, polyoxyethylenenonylphenylether;IEF, isoelectric focusing;SDS, sodium dodecyl sulfates