Purification and characterization of a liver microsomal cytochrome P-450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole-treated D2 mice.

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 selectivity 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 microM 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.