Tryptophan decarboxylase is an enzyme that catalyzes the conversion of tryptophan to tryptamine. Tryptamine is a first-reaction intermediate of indole-3-acetic acid (a type of auxin) biosynthesis.
Therefore, to study the tryptamine pathway of indole-3-acetic acid biosynthesis, it is necessary to check the activity of the enzymes involved in the pathway. Here, I have mentioned an easy protocol to check the activity of the tryptophan decarboxylase enzyme. You can increase or decrease the reaction volume with an appropriate ratio.
Take 0.1 ml of crude extract and mix with 0.3 ml of tryptamine assay buffer (0.2 mmol Tryptophan and 16 nmol pyridoxal phosphate in 0.2 M bicarbonate buffer of pH 7.5). Now incubate the reaction mixture for 1 hour at 30 ºC and stop the reaction by adding 0.4 ml of 1M KOH and extract the tryptamine with 2 ml of ethyl acetate (Islas et al., 1994).
Use the organic phase for the fluorimetric detection of tryptamine at 280 nm excitation and 340 nm emission wavelengths while you can also use the aqueous phase to calculate unreacted L-tryptophan with the help of the ninhydrin test.
For the ninhydrin test, take 0.5 ml of aqueous sample and mix with 0.5 ml of ninhydrin reagent prepared by dissolving 250 mg of ninhydrin in 10 ml of 0.6 M acetic acid and phosphoric acid (3:2 v/v) (Gaitonde and Dovey, 1970). Mix the reaction mixture and cover the test tube with a metal cap.
Heat the reaction mixture in a boiling water bath for 10 minutes. After 10 minutes, allow the test tube to cool and add 3 ml of 95 % ethanol to make the total volume 4 ml. Now, mix the content and read the absorbance at 400 nm wavelength. Don’t forget to use the reagent alone as a blank (Gaitonde and Dovey, 1970). However, to calculate the amount of unreacted L-tryptophan you have to prepare a standard calibration curve of tryptophan using known concentrations.
Reference:
- Islas I, Loyola-Vargas, VM, de Lourdes Miranda-Ham, M. 1994. Tryptophan decarboxylase activity in transformed roots from Catharanthus roseus and its relationship to tryptamine, ajmalicine, and catharanthine accumulation during the culture cycle. In Vitro Cellular & Developmental Biology-Plant 30(1):81-83.
- Gaitonde MK, Dovey T. 1970. A rapid and direct method for the quantitative determination of tryptophan in the intact protein. Biochemical Journal 117(5):907-911.
- Wang M, Li Q, Fiore SD, Fischer R. 2001. Expression of recombinant tryptophan decarboxylase in different subcellular compartments in tobacco Acta Botanica Sinica, 44(3):314-317.
