Purification and Properties of Bovine Brain Dopamine β‐Hydroxylase

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Abstract
Dopamine .beta.-hydroxylase (DBH) was purified from bovine brain by a series of steps including extraction with 0.5% Triton X-100, ammonium sulfate fractionation and serial chromatographies with concanavalin A (Con A)-Sepharose, Biogel A-1.5 m, DEAE-Sephadex and phenyl-Sepharose. The overall purification was .apprx. 4200-fold, and the final specific activity was 147 nmol/min per mg protein. Bovine brain DBH was apparently a glycoprotein and interacted with immobilized Con A. The enzyme bound to phenyl-substituted agarose by hydrophobic interaction. An approximate MW of 400,000 was estimated by gel filtration; the protein eluted earlier than bovine adrenal DBH with a MW estimated to be 290,000. The Km values toward tyramine and ascorbate were 1.53 and 1.42 mM, respectively, the optimal pH was 5.0 in the presence of 20 mM tyramine as substrate. Immunological titration studies indicated that bovine brain and adrenal DBH had common antigenic sites. A close similarity between the bovine brain and adrenal enzyme was shown.