Phenylalanine ammonia-lyase (L-phenylalanine ammonia-lyase, PAL) is an enzyme that catalyzes the direct removal of ammonia from L-phenylalanine to form trans-cinnamic acid. This enzyme is mostly found in the soluble fractions of higher plants, yeasts, and fungi. It was first discovered in barley by J. Koukol in 1961, with an estimated molecular weight of approximately 300,000 Da. As a key enzyme that channels L-phenylalanine into the synthesis of phenolic compounds, its activity in tissues can undergo significant changes in response to external factors. Treatments such as light exposure, pathogen infection, wounding, and application of plant hormones can markedly enhance its activity. In most cases, a subsequent inactivation process occurs following the elevation of PAL activity in tissues, leading to a rapid decline in the amount of active enzyme. This inactivation is believed to be associated with the action of protein-like substances.
The Phenylalanine Ammonia-Lyase (PAL) Extraction Reagent is primarily designed for lysing plant tissues and extracting PAL from samples. This reagent is intended for research use only and is not suitable for clinical diagnosis or any other purposes.
Materials to Be Prepared by the User
1. Distilled water
2. Centrifuge tubes or test tubes, homogenizer or mortar and pestle, refrigerated centrifuge
Operating Procedures (For Reference Only)
1. Take the plant tissue, rinse it thoroughly, and cut it into small pieces.
2. Add the PAL Extraction Reagent at a ratio of 2 g plant tissue: 5 ml reagent, then fully homogenize or grind the mixture under ice bath conditions.
3. Centrifuge at 10,000×g and 4°C for 15-20 minutes, and collect the supernatant.
4. Store the supernatant at -20°C for subsequent PAL activity assays or other experimental applications.
Calculation
Yield of crude enzyme solution from samples (ml/g)=Volume of supernatant (ml)/Mass of sample (g)×100%
Precautions
1. The sample to be tested must not contain phosphatase inhibitors, and repeated freeze-thaw cycles should be avoided.
2. If the measured value of the sample exceeds the upper limit of the standard curve, dilute the sample with the extraction reagent and perform the assay again.
3. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
4. For your safety and health, wear a lab coat and disposable gloves during the operation.
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