Determination of phenylalanine ammonia-lyase (PAL) activity

The basic principle behind the determination of phenylalanine ammonia-lyase (PAL) activity is that phenylalanine ammonia-lyase is a key enzyme in plant secondary metabolism. It catalyzes the deamination of L-phenylalanine. Ammonia is released to form trans-cinnamic acid. The activity of the enzyme can be determined from the change in absorbance of the product, trans-cinnamic acid, at a wavelength of 290 nm.

Operation method

Determination of phenylalanine ammonia-lyase (PAL) activity

Principle

The basic principle behind the determination of phenylalanine ammonia-lyase (PAL) activity is that phenylalanine ammonia-lyase is a key enzyme in plant secondary metabolism. It catalyzes the deamination of L-phenylalanine. Ammonia is released to form trans-cinnamic acid. The activity of the enzyme can be determined from the change in absorbance of the product, trans-cinnamic acid, at a wavelength of 290 nm.

Materials and Instruments

Material: yellowing rice seedlings.
Equipments: freezing high-speed centrifuge, UV-visible spectrophotometer, vortex mixer, constant temperature water bath.
Reagents:
①0. 1 mol ・L
-①0. 1 mol ・L
Boric acid buffer (pH 8.8)
② 0.02 mol ・ L
L-phenylalanine: Weigh 3.33 g of L-phenylalanine.
L-phenylalanine: Weigh 3.33 g of L-phenylalanine and dissolve it in 1 000 mL of 0. 1 mol ・L -1 borate buffer (pH 8. 8).
L-1
L-phenylalanine: Weigh 3.33 g of L-phenylalanine and dissolve it in 1,000 mL of 0.1 mol・L-1 borate buffer (pH 8.8).
③7 mmol - mercaptoethanol borate buffer: 0. 11 mL of contemptuous ethanol was dissolved in 0. 1 mol・L -1 borate buffer (pH 8. 8) with 0. 1 mol・L -1 borate buffer (pH 8. 8).
-1
Boric acid buffer: 0.11 mL of 0.1 mol・L-1 ethanol was dissolved in 0.1 mol・L-1 borate buffer, and the volume was fixed to 200 mL
④Polyvinylvinylpyrrolidone (PVP)

Move

The basic procedure for the determination of phenylalanine ammonia-lyase (PAL) activity can be divided into the following steps:

1. Enzyme solution preparation: Weigh 0.5 g of yellowing rice seedlings, add 1.5 mL of pre-cooled extraction solution (i.e., 7 mmol ・ ^L-1 Vegetable Ethanol Borate Buffer), excessive polyvinylpyrrolidone (PVPX) (but not too much, otherwise it is not easy to be ground), and a small amount of quartz sand to make a slurry under an ice bath, and then add 3.5 mL of pre-cooled extract to make a final volume of 5 mL. Centrifuge at 12,000 g for 15 min at 4°C, and suck the supernatant with a pipette, i.e. the crude enzyme solution. 2.

2. Enzyme activity was determined as follows:
The reaction solution consisted of: ① 0.02 ^mol・L-1 L-phenylalanine 1 mL;
② 0.1 ^mol・L-1 boric acid buffer (pH 8.8) 2 mL;
③ 0.1 mL of crude enzyme solution.
(For control, 0.1 mL of ethanol sparing buffer was used instead of the enzyme solution.)
The reaction solution was mixed in a vortex mixer and the starting absorbance value was measured at 290 nm immediately after mixing and timed accurately. （The tubes were kept in a water bath at 30°C for 30 min, and then the absorbance values of each tube were measured at 290 nm. The amount of enzyme required to increase the absorbance at 290 nm by 0.01 per 30 min was expressed as one unit (U).
Phenylalanine deaminase activity (U - ^g-1 fresh weight) =
where a - amount of enzyme solution used in the assay, mL;
V - total volume of enzyme solution, mL;
W - weight of sample, g.
(Phenylalanine deaminase activity can also be expressed as U - ^mg-1 protein. (Protein content can be determined by the G-250 method using bovine serum albumin as the standard).

Caveat

Requires the spectrophotometer to have an accurate wavelength.

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