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In the adrenal cortex, NADPH-adrenal ferredoxin reductase (adrenodoxin reductase)-adrenal ferredoxin (adrenodoxin)-cytochrome P-450 system has been known to be associated with the mitochondrial steroid hydroxylase activities, viz., cholesterol side-chain cleavage and steroid 11β- and 18-hydroxylations. In order to elucidate the reaction mechanism of the hydroxylase system, it is important that each component is available in a pure form. As previously reported, adrenodoxin reductase (1) and adrenodoxin (2,3) have been obtained in a pure state. The latter has been crystallized. Details will be discussed in a separate chapter (4).

Native and reconstituted adrenal ferredoxins have been obtained in crystalline form. The apoprotein was prepared by treatment of the native protein with trichloroacetic acid. When the apoprotein was incubated with ferrous ion, sulfide and 2-mercaptoethanol, the recovery of the reconstituted protein was considerably low. The reconstitution was greatly enhanced by the presence of 8 M urea. The reconstituted protein was indistinguishable from the native protein with respect to enzymic activity, spectral properties and iron content. The apparent contents of labile sulfide in both native and reconstituted proteins were about one mole per mole of protein when analyzed according to the original methylene blue method without alkaline zinc incubation. Extension of the alkaline zinc incubation over 2 hr resulted in release of two moles of labile sulfide per mole of protein.