A Sensitive and Reliable Assay for Dopamine (β‐Hydroxylase in Tissue

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Abstract
A new assay procedure for dopamine .beta.-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on concanavalin A-sepharose (Con A-sepharose), resulting in enrichment of the enzyme and removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-sepharose. The adsorption of the DBH to Con A-sepharose offers 3 advantages over previous assay procedures. Because of removal of the endogenous inhibitory substances, a single Cu2+ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analyzed sample. Using this procedure, the optimal Cu2+ concentration for DBH of bovine adrenal gland extracts was 3 .mu.M and for rat brain 10 .mu.M. Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline [norepinephrine], was determined by high performance liquid chromatography and electrochemical detection. This procedure resulted in an .apprx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. The Km for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.