Assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase based on rapid separation of the reaction product by high performance liquid chromatography.
A rapid separation of 5-hydroxytryptophan by high performance liquid chromatography (HPLC) was achieved for the assay of tryptophan hydroxylase. "Bulk separation" of the product from all other components in the reaction mixture by HPLC was achieved by 1) the choice of a suitable column-solvent system so as to elute the reaction product ahead of other components in the sample mixture, 2) the use of a monitor selective for the reaction product, 3) minimization of the column length so as to achieve rapid separation of the product from the substrate. The method finally employed a reversed phase column of 5 cm length, relatively rapid elution at 2 ml/min and fluorescence detection at 350 nm with an excitation at 302 nm. The assay is convenient and as sensitive as the radioisotope method. The advantages of the method are 1) almost no pretreatment of samples, 2) repeatability every 2 min, 3) wide latitude of product determination from picomole to nanomole amounts per assay. The method was extended to the assay of 5-hydroxytryptophan decarboxylase by essentially the same procedures.
D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007700:Kinetics; D012515:Mast-Cell Sarcoma / Q000201:enzymology; D051379:Mice; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014365:Tryptophan Hydroxylase / Q000032:analysis* / Q000378:metabolism
D014365:Tryptophan Hydroxylase; D001142:Aromatic-L-Amino-Acid Decarboxylases