Simultaneous HPLC Analysis of Tryptophan Hydroxylase Activity and Serotonin Metabolism in Rat Pineal Gland: Determination of Its Kinetic Properties

摘要

The determination of tryptophan hydroxylase activity (TPH) in the pineal gland was based upon the separation and detection of 5-hydroxy tryptophan (5OH-TRP) formed from L-Tryptophan (TRP) by high performance-liquid chromatography (HPLC) with fluorimetric detection. Tryptophan hydroxylase activity and TRP metabolites content show circadian variation in the rat pineal gland. The enzyme exhibited a Km value of 53 ± 15 μM and a Vmax of 243 ± 23 pmol 5OH-TRP/min/mg. prot. for L-TRP, and a Km value of 27 ± 4.54 μM and a Vmax of 90.2 ± 4.35 pmol 5OH-TRP/min/mg. prot. for tetrahydrobiopterine. The present assay is accurate, simple and sensitive, allowing the determination of TPH activity in a variety of enzyme sources. The combination of simultaneous measurements of serotonin, as well as serotonin precursors and metabolites, from a single tissue sample makes it extremely useful for physiological and pharmacological studies.