Direct fluorometry of phase-extracted tryptamine-based fast quantitative assay of L-tryptophan decarboxylase from Catharanthus roseus leaf

Analytical Biochemistry
R S SangwanS Kumar

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 > or = 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 microM tryptamine. The examination of interassay variations and the comparative magnitude of fluorescence response allowed to infer that a satisfactory and sufficient sample luminesce...Continue Reading

References

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Citations

Feb 13, 2008·The Plant Journal : for Cell and Molecular Biology·Atsushi IshiharaKyo Wakasa
Jan 24, 2019·Plant Biotechnology Journal·Thomas RademacherJohannes Buyel
Feb 19, 2013·The Protein Journal·Sangita Dutta, Debasish Bhattacharyya
Apr 13, 2018·Reproductive Biology and Endocrinology : RB&E·Nirja ChaudhariLaxmipriya Nampoothiri

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