Methodology for Enzymes

Phenylalanine Ammonia Lyase
(L-Phenylalanine Ammonia Lyase EC 4.3.1.5)

Phenylalanine ammonia lyase (PAL) is responsible for the conversion of L-Phenylalanine to trans-cinnamic acid.

Cinnamic acid serves as a precursor for the biosynthesis of coumarins, isoflavanoids and lignin. These compounds play an important role in pest and disease resistance mechanism. Changes in PAL activity accompanying fungal, viral and bacterial infections of plants have been reported.





Principle
Phenylalanine ammonia lyase activity is determined spectrophotometrically by following the formation of transcinnamic acid which exhibits an increase in absorbance at 290nm (crude enzyme)/270nm (purified enzyme).





Materials
» Borate Buffer 0.2M (pH 8.7)

» L-Phenylalanine 0.1M
Dissolve 165mg L-Phenylalanine in 10mL water and adjust to pH 8.7 with 0.1NKOH.

» 1M Trichloro Acetic Acid
Dissolve 16.3g in 100mL water.

» Enzyme Extract
Homogenize 500mg of the plant material in 5mL of cold 25mM borate-HCl buffer pH 8.8 containing 5mM mercaptoethanol (0.4mL/L). Centrifuge the homogenate at 12,000g for 20min. Use the supernatant as enzyme source.


Procedure
1.
Pipette out 0.5mL borate buffer, 0.2mL enzyme solution and 1.3mL water in a test tube.
2.
Initiate the reaction by the addition of 1mL L-Phenylalanine solution.
3.
Incubate for 30-60 min at 32°C.
4.
Stop the reaction by the addition of 0.5mL of 1M trichloroacetic acid.
5.
Run a control in which add phenylalanine after trichloroacetic acid.
6.
Measure the absorbance at 290nm.
7.
Prepare a standard graph for trans-cinnamic acid.


Calculation
Express the reaction rate as micromole trans-cirmdimic acid formed per mg protein per min.



References
1.  Brueske, C H (1980) Physiol Plant Pathol 16 409.