A simple and sensitive fluorometric assay for tyrosine hydroxylase

✍ Scribed by Takashi Yamauchi; Hitoshi Fujisawa

• Publisher: Elsevier Science
• Year: 1978
• Tongue: English
• Weight: 419 KB
• Volume: 89
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(78)90735-2

✦ Synopsis

A simple, rapid, and sensitive fluorometric assay for measuring the activity of tyrosine hydroxylase is described. The enzyme activity is detected by converting tyrosine to 3,4-dihydroxyphenylalanine (dopa), which is then subjected to conversion to the highly fluorescent product by the trihydroxyindole method. The assay method is very reproducible, more sensitive than a radiochemical method for the determination of tyrosine hydroxylase activity using the isolation of [3H]water commonly used, and linear from 0.2 to 12 nmol of dopa. The method should be applicable for the assay of the enzyme with a wide range of activity.

Tyrosine hydroxylase (E.C. 1.14.16.2) catalyzes the conversion of tyrosine to dopa (l), the rate-limiting step in the biosynthesis of catecholamines (2). The recent development of sensitive radiochemical assay methods (1,3,4) for tyrosine hydroxylase has contributed substantially to an understanding of the regulation of the biosynthesis of catecholamines. The fluorometric method described in this communication does not require the use of radioactive materials or a liquid scintillation spectrometer and, furthermore, provides the sensitivity and reproducibility comparable with those obtained with radiochemical methods.

MATERIALS AND METHODS

Materials.

L-[3,S3H]Tyrosine (1 Ci/mmol) was obtained from Amersham Radiochemical

Center and was purified according to the method of Ikeda et al. (5). L-[lJ4C]Tyrosine (53.6 mCi/mmol) and DL-[lJ4C]dopa (13.2 mCi/mmol) were obtained from New England Nuclear Corp. 6-MPH, and DMPH4 were purchased from Calbiochem. Crystalline catalase was obtained from Boehringer-Mannheim Corp. Alumina (Aluminium oxide