Cytochrome mammalian, purification

Multiplicity of Cytochrome P450, Purification, and Reconstitution of Cytochrome P450 Activity. Even before appreciable purification of CYP had been accomplished, it was apparent from indirect evidence that mammalian liver cells contained more than one CYP enzyme. Subsequent direct evidence on the multiplicity of CYPs included the separation and purification of CYP isozymes, distinguished from each other by chromatographic behavior, immunologic specificity, and/or substrate specificity after reconstitution and separation of distinct polypeptides by sodium... 

Although mammalian CYPs are attractive candidates for use as commercial biocatalysts, many functional characteristics limit the opportunities to exploit such a system. Association of the enzymes with membranes prevents easy extraction and purification and limits the opportunities to produce useful recombinant enzymes by cloning the relevant genes for expression in microbial systems. All P450s have a porphyrin-haem active site that requires a second protein to reduce the iron component, often cytochrome P450 reductase or... 

As noted above, cytochrome b in the mammalian respiratory system is firmly bound to the mitochondrial membrane and cannot be solubilized without using detergents such as cholate. It is thus interesting that a cytochrome b-like pigment, cytochrome b-563, is easily extracted from the larvae and pupae of the housefly, Musca domestica, with a simple salt solution, but it cannot be extracted from the adult flies under the same conditions. The cytochrome is obtained as a crystalline preparation after purification by the chromatography on DEAE-cellulose (78,84). The crystalline cytochrome b-563 has a similar spectral property to mammalian cytochrome b and shows absorption peaks at 563, 530, and 428.5 nm in the reduced state. Contrary to purified mammalian cytochrome b, this cytochrome does not combine with carbon monoxide and exists as monomer species in the solution without detergent. 

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