Phenylalanine deaminase (PAL)
Phenylalanine ammonia-dissolving enzyme (PAD) plays an important role in regulating the formation of secondary substances such as lignin, phytoalexins, flavonoids and anthocyanins in plants, and it is also related to the disease resistance of plants. This experiment is to understand the principles and methods of measuring PAD activity.
Principle
Phenylalanine deaminase 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 290 nm.
Operation method
Phenylalanine deaminase (PAL)
Principle
Phenylalanine deaminase 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 290 nm.
Materials and Instruments
1 . Materials: yellowing rice seedlings. Move 1. Enzyme solution preparation: Weigh 0.5 g of yellowing rice seedlings, add 1.5 mL of pre-cooled extract (i.e., 7 mmol-L-1 Vegetable Ethanol Boric Acid Buffer), excessive polyvinylpyrrolidone (PVPX, but not too much, otherwise it is not easy to be grinded), and a small amount of quartz sand to form a slurry under an ice bath, and add 3.5 mL of pre-cooled extract to make the final volume 5 mL. centrifugation was carried out at 12,000 g for 15 min at 4°C, and the supernatant was aspirated with a pipette, i.e., crude enzyme solution. Centrifuge at 12 000 g for 15 min at 4° C. Pipette the supernatant, i.e. the crude enzyme solution. 2. Caveat Requires the spectrophotometer to have an accurate wavelength. Common Problems How does phenylalanine deaminase relate to disease resistance in plants? For more product details, please visit Aladdin Scientific website.
2. Reagents: 0.1 mol-L] boric acid buffer (pH 8.8); 0.02 mol-L 1 L-phenylalanine: 3.33 g of L-phenylalanine was weighed and dissolved in 1,000 mL of 0.1 mol-L "1 boric acid buffer (pH 8.8); 7 mmol - tungsti-ethanol borate buffer: 0.11 mL of tungsti-ethanol was dissolved in 0.1 mol-L-1 borate buffer, and the volume was set to 200 mL. 0.11 mL of ethanol was dissolved in 0.1 mol.L-1 boric acid buffer, and the volume was fixed to 200 mL; polyvinylpyrrolidone (PVP).
3 . Equipment: freezing high-speed centrifuge, UV-visible spectrophotometer, vortex mixer, constant temperature water bath.
2. Enzyme activity measurement:
The reaction solution consists of: ① 0.02 mol - L_1 L-phenylalanine 1 mL;
① 0.02 mol - L_1 L-phenylalanine 1 mL; ② 0.1 mol - L 1 boric acid buffer (pH 8.8) 2 mL;
(iii) 0.1 mL of crude enzyme solution.
(For control, 0.1 mL of sparse ethanol buffer was used instead of enzyme solution.)
Immediately after mixing the reaction solution with a vortex mixer, the starting absorbance value was measured at 290 nm and accurately timed. （The tubes were kept in a water bath at 30 °C for 30 min, and the absorbance values of the tubes 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 ammonia-lyase activity (U - g 'fresh weight) = 3. min 2 就鷲 ［X V
a A W X U. U1
where s 一measurement of enzyme solution, mL;
V-total volume of enzyme solution, mL;
W-sample weight, g.
(Phenylalanine ammonia-lyase activity can also be expressed in terms of U - mg" 1 protein). Protein content can be expressed as U - mg" 1 protein. (Protein content can be determined by the G-250 method using bovine serum albumin as the standard).