It was suggested by Blaschko (1) that the rate-limiting step in the biosynthesis of the pressor amines is the hydroxylation of the side chain of 3, 4-dihydroxyphenylethylamine to form norepinephrine. The reaction has been demonstrated in vitro with the use of adrenal slices and homogenates (2, 3) and aqueous extracts of adrenal acetone powder (4, 5), but little has been learned about the mechanism of the hydroxylation. In homogenates, the enzymatic activity is located in the particulate fraction obtained by high speed centrifugation(6), and until recently large amounts of activity have not been obtained in a soluble form. The restrictions which this has placed on purification of the enzyme system have limited detailed studies of the reaction mechanism.

It was recently reported that the hydroxylating enzyme can be solubilized from particles of beef adrenal medulla (7). The present report describes the details of the solubilization method and procedures for the further purification of the enzyme. With the use of this partially purified enzyme preparation, a requirement for ascorbic acid and oxygen has been observed. Balance studies indicate that in the reaction catalyzed by this enzyme 1 mole of ascorbate is oxidized to dehydroascorbate for each mole of DOPaminel converted to norepinephrine. In support of this formulation, a DOPamine-dependent oxidation of ascorbate has been demonstrated. A relationship thus exists between this system and the system catalyzing the hydroxylation of the aromatic ring of phenylalanine: in each case hydroxylation of the substrate is coupled to oxidation of an equimolar amount of a cofactor (8).