Chromatographic separation and behavior of microsomal cytochrome P450 and cytochrome b5

The methods used for separation of the multiple mammalian cytochrome P450 enzymes by liquid chromatography are reviewed. In addition to the chromatographic techniques, preparation and handling of samples and prefractionation procedures are considered. Conditions that affect stability and chromatographic resolution of cytochromes P450 are also discussed. Special emphasis is put on useful methods which are not routinely used for P450 separation, such as immobilized metal affinity or hydrophobic-interaction chromatography. Applications of low- and high-pressure methods with regard to preparative and analytical separations are compared. It is shown that high- and medium-pressure ion-exchange chromatography are suitable tools for separation of closely related P450 enzymes, especially when specific detection methods are available. In addition to fractionation of cytochromes P450, the isolation and chromatographic behavior of cytochrome b5 is discussed.