Direct fluorometry of Phase-extracted tryptamine-based fast quantitative assay of L-tryptophan decarboxylase from Catharanthus roseusleaf

Abstract

An assay for the enzyme l-tryptophan decarboxylase (TDC; EC 4.1.1.28) is described. It is based on direct fluorometry of the enzymatic reaction product (tryptamine) selectively recovered in ethyl acetate from the reaction mixture. Catalytically formed tryptamine from tryptophan in the incubation mixture is selectively (free from tryptophan) physically separated as ethyl acetate solution under basic (pH ≥ 11) conditions and subjected to direct fluorescence measurement in the organic solvent using a spectrofluorometer with excitation and emission wavelengths of 280 and 350 nm, respectively. Tryptamine production rate was quantitated from the luminescence response curve of tryptamine drawn under similar extraction and measurement conditions. Luminescence calibration curves were drawn for tryptamine in aqueous (water or buffer system) as well as in organic solvent as recovered from the varied aqueous solution conditions including those similar to the enzyme incubation mixture. The luminescence calibration graphs were linear for at least 0.5 to 10 μM tryptamine. The examination of interassay variations and the comparative magnitude of fluorescence response allowed to infer that a satisfactory and sufficient sample luminescence response was retained under the varied conditions including those akin to the enzymatic assay mixture, allowing adaptation of the fluorometry for the TDC activity quantitation. The assay was found to follow the proportionality principle of product formation with respect to catalytic reaction time as well as protein concentration in the assay mixture using Catharanthus roseusleaf crude homogenate as well as the enzyme preparation at different states of purity. The rate of tryptamine formation under the catalytic conditions was linear for at least 1 h at 30°C. Though the assay has been demonstrated to use the C. roseusleaf as the enzyme source, it should be equally applicable to other plant and nonplant sources. The merits and precautions of the protocol have been discussed.