Abstract
As a model for the assay of enzyme activity by high-performance liquid chromatography, a simple, sensitive and specific fluorescence assay for dopamine β-hydroxylase (DBH) activity was devised using o-phthalaldehyde as reagent. Tyramine was used as a substrate and was incubated under optimal conditions. Endogenous inhibitors in the enzyme preparation were completely inactivated by addition of N-ethylmaleimide and a small amount of Cu 2+. After isolating the amines on a small Dowex 50 column, the eluate was analyzed fluorometrically by high-performance liquid chromatography. The product, octopamine, was eluted prior to and completely separated from tyramine, and the height of the fluorescence peak gave the amount of enzymatically formed octopamine. DBH activity in serum obtained from the umbilical vein was much lower than the activity in adult human serum. Normal values of serum DBH activity from 153 subjects (1–74 years) showed a mean and standard deviation of 42.5 ± 30.9 and 34.3 ± 18.2 μmol/min/liter of serum at 37°C, before and after computation by the method of Hoffmann. Male serum had a slightly higher activity than female serum. Serum DBH activity rapidly increased during the first 10 years after birth, gradually increased up to an optimum at 40–50 years, and then decreased.
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