A rapid and sensitive fluorescence assay for angiotensin-converting enzyme.

A new, highly sensitive, specific assay for dopamine-β-hydroxylase (DBH) activity in human serum is described. Tyramine is used as a substrate; the product of the enzymatic hydroxylation, octopamine, is converted by reacting with 1-dimethylaminonaphthalene-5-sulfonyl-chloride (Dns-Cl) to a fluorescent product, which is extracted from the reaction mixture and purified from the extract by thin-layer chromatography (tlc). The fluorescence of the dansylated octopamine is measured in situ on the tlc plate using a chromatogram-spectrofluorometer. This one-step enzyme reaction can be performed at optimum pH and substrate concentration. As little as 8 ng of octopamine can be determined accurately; the response is linear up to more than 400 ng of octopamine. A comparison with the radioenzymatic assay (Weinshilboum, R., and Axelrod, J. (1971) Circ. Res.28, 307–315) shows an approximately twofold increase in the enzymatic activity measured. Kinetic studies of human sera with high and low DBH activity gave a K_m value of 3.1 × 10^?3m. The method is successfully being used for the functional characterization of the enzyme and genetic studies (Herschel, M., in preparation).