Open AccessPurification and characterization of a liver microsomal cytochrome P ‐450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole‐treated D2 mice
Author(s) -
JUVONEN Risto O.,
SHKUMATOV Vladimir M.,
LANG Matti A.
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb13777.x
Subject(s) - coumarin , pyrazole , microsome , isozyme , cytochrome , chemistry , cytochrome p450 , biochemistry , stereochemistry , metabolism , enzyme , organic chemistry
Microsomal coumarin 7‐hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612–614 ]. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J. Biochem. 152 , 3–8]. This paper describes the purification of the pyrazole‐inducible cytochrome P‐450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion‐exchange chromatography and the presence of 100 μM coumarin in the preparations throughout the whole purification. Coumarin effectively protected the P‐450 from degradation and also converted the pyrazole‐inducible P‐450 to its high‐spin state. This enabled us to choose only those fractions for further purification where the P‐450(s) was in its high‐spin state (rather than measuring the content of the total P‐450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.