Assay for Tryptophan Hydroxylase Activity in Rat Brain by High-Performance Liquid Chromatography with Fluorescence Detection

• Published in: Biological & pharmaceutical bulletin Vol. 19; no. 5; pp. 762 - 764
• Main Authors: IIZUKA, Ryuji, ISHIDA, Junichi, YOSHITAKE, Takashi, NAKAMURA, Masaru, YAMAGUCHI, Masatoshi
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.05.1996; Maruzen; Japan Science and Technology Agency
• Subjects: 5-hydroxyindole; 5-Hydroxytryptophan - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Brain - enzymology; Chromatography, High Pressure Liquid - methods; Enzymes and enzyme inhibitors; fluorescence; Fundamental and applied biological sciences. Psychology; HPLC; Male; Oxidoreductases; rat brain; Rats; Rats, Wistar; Spectrometry, Fluorescence; tryptophan 5-monooxygenase; Tryptophan Hydroxylase - metabolism; Fluorescence detector; Rat; Enzyme; Rodentia; Central nervous system; HPLC chromatography; Vertebrata; Mammalia; Enzymatic activity; Tryptophan 5-monooxygenase; Animal; Oxidoreductases; Measurement method; Brain (vertebrata)
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.19.762

A highly sensitive and selective liquid chromatographic (HPLC) method with post-column fluorescence detection has been developed for the determination of tryptophan 5-monooxygenase activity in rat brain tissue homogenate. 5-Hydroxytryptophan, formed enzymatically from tryptophan (incubation time, 20 min), is extracted with perchloric acid and determined by HPLC. Detection is performed fluorometrically after post-column derivatization by a reaction with benzylamine in the presence of potassium hexacyanoferrate (III). The lower limit of detection for 5-hydroxytryptophan formed enzymatically is 100 fmol at a signal-to-noise ratio of three.