A SENSITIVE ASSAY FOR DOPAMINES‐β‐HYDROXYLASE

A sensitive procedure for the determination of dopamine‐β‐hydroxylase (EC 1.14.2.1) activity in homogenates of rat brain and in rat serum is described. In the assay, the substrate, [14C]tyramine is enzymatically converted to [14C]octopamine which is then oxidized with periodate to the [l4C]p‐hydroxybenzaldehyde. The latter compound is separated by solvent extraction into ether and its radioactivity determined. A simple method has been developed for the purification and convenient storage of [14C]tyramine which results in a boiled‐blank value of about 100 d.p.m. per 106 d.p.m. of [I4C]tyramine. The low blank allows the detection of as little as 7.5 pmol of product. This makes the procedure several times more sensitive than other methods now available. The interactions of copper, N‐ethylmaleimide and p‐chloromercuriphenyl sulfonic acid with the endogenous inhibitors were also examined. The method should be generally applicable for the assay of DBH in any tissue homogenate once the appropriate copper and dilution parameters have been determined.