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Assay for tryptophan hydroxylase activity in rat brain by high-performance liquid chromatography with fluorescence
R Iizuka1, J Ishida, T Yoshitake
1Chemical Biotesting Center, Chemical Inspection and Testing Institute, Oita, Japan.
Abstract:
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.