Determination of dopamine-β-hydroxylase activity by means of chromatogram-spectrofluorometric measurement in remission
✍ Scribed by Marijana Kopun; Michael Herschel
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 427 KB
- Volume
- 85
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A new, highly sensitive, specific assay for dopamine-P-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 I-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 (tic). The fluorescence of the dansylated octopamine is measured in situ on the tic 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][308][309][310][311][312][313][314][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, value of 3.1 x 10m3 M. The method is successfully being used for the functional characterization of the enzyme and genetic studies (Herschel, M., in preparation).