Experimentally, the assay of polyphenol oxidase activity in plants was mastered.

Operation method

Experiments for the determination of polyphenol oxidase activity in plants

Principle

Polyphenol oxidase is a copper-containing oxidase that oxidizes mono- and di-phenols to produce quinones in the presence of oxygen. The activity and specific activity of polyphenol oxidase can be calculated by measuring its absorbance spectrophotometrically at 525 nm. The reaction formula is as follows: .

Materials and Instruments

Potato tubers
Polyvinylpyrrolidone Phosphoric acid buffer Phosphoric acid buffer Haldol ammonium sulfate Catechol Trichloroacetic acid
UV-1206 UV-1240 Spectrophotometer Centrifuge Constant temperature water bath Mortar or homogenizer Test tubes Pipettes Gauze bags

Move

1. Preparation of crude enzyme solution

Weigh 5g of potato tubers in a mortar and pestle, add 0.5g of insoluble polyvinylpyrrolidone (previously soaked in distilled water, and then filtered to remove impurities) and 100 ml of 0.1 mol-L-1pH6.5 phosphate buffer, grinding into a homogenate, filtered through several layers of gauze bag, filtrate added 30% saturation ammonium sulfate, centrifugation to remove the precipitate, and then add ammonium sulfate to reach 60% saturation, and then collect the precipitate by centrifugation. The supernatant was then saturated with 60% ammonium sulfate, and the precipitate was collected by centrifugation. The precipitate was dissolved in 2~3 ml of 0.01mol-L-1 pH6.5 phosphate buffer, i.e. the crude enzyme solution.

2. Determination of active enzyme

Add 3.9 ml 0.05 mol-L-1 pH5.5 phosphate buffer, 1.0 ml 0.1 mol-L-1 catechol in a test tube and keep warm for 10 min at 37℃ in a constant temperature water bath, and then add 0.5 ml enzyme solution (depending on the enzyme activity to increase or decrease the amount of), shake quickly, pour into the colorimetric cup, and measure at 525nm. The absorbance change (A) was measured at 525 nm with time scanning in 1~2 min.

Common Problems

Calculation of enzyme activity:

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