PPO activity assay can be applied to (1) study the physiological mechanism and regulation of PPO. (2) To understand the relationship between enzyme activity and browning of plant tissues and physiological activities.
Operation method
spectrophotometry
Principle
PPO catalyzes the oxidation of various phenols with O2 to quinone. In this experiment, catechol was used as a substrate and PPO catalyzed the formation of brown quinone from catechol in a reaction system of 0.2M disodium hydrogen phosphate-0.1M citric acid buffer pH 5.8, which caused a change in the OD value of the reaction system at 410 nm on a spectrophotometer, and the magnitude of the enzyme activity of PPO was determined by the change in the OD rise reading.
Materials and Instruments
Polyphenol oxidase crude enzyme solution Move In a test tube containing 4 ml of 0.2 M disodium hydrogen phosphate-0.1 M citrate buffer of pH 5.8, 0.05 ml of 0.05 M catechol solution was added and 0.05 ml of enzyme solution was added after preheating in a thermostatic water bath at 30°C. The reaction was carried out for 5 min and absorbance values were read at 410 nm on a spectrophotometer. One unit of enzyme activity (U) was defined as an increase of 0.01 O.D. value per minute under the above conditions by reading at A410. Caveat 1. The reaction mixture must be prepared and used now, otherwise it will be invalid due to the auto-oxidation of catechol;2. When adding samples, add the buffer solution first, then add the substrate catechol, shake well, and add the enzyme extract last before measurement.3. A phenol binding agent, polyvinylpyrrolidone (PVPP), is added to the test, which can strongly bind with phenolic compounds, thus eliminating the substrate in the enzyme reaction system and making the measurement of PPO activity closer to its true value. Common Problems The relationship between PPO and disease resistance has been extensively studied. Resistance-associated enzymes play an important role in plant defense against pathogenic microorganisms, which mainly include some enzymes in the phenolic metabolic system and the pathogen-associated protein family PPO can protect cells from pathogens by catalyzing the formation of lignin and quinone compounds, which constitutes protective shielding, or it can play a direct role in resistance through the formation of quinone substances. Since a large number of polyphenols with different structures exist in nature, the polyphenol oxidases that catalyze the oxidation of these phenols are also different. It would be of far-reaching significance if effective enzyme sources are screened from microorganisms or created by using techniques such as enzyme modification, gene heterologous expression and construction of genetically engineered bacteria. For more product details, please visit Aladdin Scientific website.
Catechol solution Citrate buffer Sodium dihydrogen ophthalate Ammonium sulfate precipitation fraction DE-52 elution fraction Dextran gel elution fraction
Test tube Constant temperature water bath Spectrophotometer