They should know the role of ascorbate peroxidase (AsA-POD). They should know how to determine ascorbate peroxidase activity spectrophotometrically.

Principle

The basic principle of ascorbate peroxidase (AsA-POD) activity is that AsA-POD catalyzes the reaction of AsA with _H2O2, which oxidizes AsA to monodehydroascorbic acid (MDAsA), and as AsA is oxidized, the extinction value in solution at 290 nm (man 29.) decreases. As AsA is oxidized, the extinction value at 290 nm (man 29) decreases, and the AsA-POD activity is calculated from the decrease in man 29 per unit time.The amount of AsA oxidized is calculated with an extinction coefficient of 2.8 (mmol-IL-cm_"), and the enzyme activity can be expressed as the amount of AsA oxidized per hour per gram of fresh weight (Mmol - gf・h^).

Operation method

Determination of Ascorbic Acid Peroxidase (AsA-POD) Activity

Principle

The basic principle of Ascorbic acid peroxidase (AsA-POD) activity is that AsA-POD catalyzes the reaction between AsA and H2O2 to oxidize AsA to monodehydroascorbic acid (MDAsA), and as AsA is oxidized, the extinction value (A290) in solution at 290 nm decreases. As AsA is oxidized, the extinction value (A290) at 290 nm decreases, and the AsA-POD activity is calculated from the decrease in A290 per unit of time.The amount of AsA oxidized is calculated by the extinction coefficient of 2.8 (mmol・L-1・cm-1), and the enzyme activity can be expressed as the amount of AsA oxidized per hour per gram of fresh weight (μmol ・g-1・h-1).The enzyme activity is calculated by the extinction coefficient of 2.8 (mmol・L-1・cm-1). The enzyme activity can be expressed as the amount of AsA oxidized per hour per gram of fresh weight (µmol ・g-1・h-1).

Materials and Instruments

Materials: fresh fruits, vegetables, plant roots, stems, leaves, etc.
Equipment: centrifuge, UV spectrophotometer, mortar, test tubes, etc.
Reagents:
①K
2
HPO
4
-KH
2
PO
4
buffer (pH 7.0, containing 0.1 mmol ・ L-1 EDTA-Na
2
)
②0.3 mmol - L
-L
AsA
③20 μmol - L
L -1 AsA ③20 μmol - L -1 AsA
L -1 AsA ③20 μmol - L -1 AsA
④0.06 mmol - L
AsA ③20 μmol - L -1 AsA ④0.06 mmol - L
H
2
O
2

Move

The basic procedure for the determination of ascorbate peroxidase (AsA-POD) activity can be divided into several steps:

1. Preparation of enzyme solution: 1.0 g of plant leaves was cut and extracted by adding pre-cooled 50 mmol・L1 _{K2 HPO4-KH2PO4} buffer at the ratio of 1:3 (W/V), then filtered through two layers of gauze, and the filtrate was centrifuged for 10 min at 4,000 r - min'1, and the supernatant was used as the crude enzyme extract for the assay.

2. 酶活性测定：3 mL 反应混合液中含 50 mmol • ^L-1, _{K2HPO4-KH2PO4} 缓冲液 (pH7.0),0.l mmol • ^L-1 EDTA- _Na2， 0. 3 mmol • ^L-1 AsA, 0. 06 0.3 mmol - L-1 AsA, 0.06 mmol - ^L-1_{H2O2 and} 0.1 mL of enzyme solution. The change of A290 was measured at 20°C for 10-30 s immediately after the addition of _H2O2, and the reduction of AsA and enzyme activity per unit of time were calculated, and the amount of enzyme reduced by 0.01 in 1 min was regarded as one unit of enzyme activity (U).

Ascorbic acid peroxidase activity [U・(g・ min)t] = WUoTl X t
where: _AA290 - change in absorbance during the reaction time;
Vt - total volume of crude enzyme extract, mL;
_V1 - volume of crude enzyme extract used for the determination, mL;
FW FW - fresh weight of the sample, g;
0. _01-A290 Every 0.01 decrease in enzyme activity is 1 unit (U);
t - reaction time, min.

Caveat

1. The extraction process of the enzyme solution should be carried out at as low a temperature as possible.

2. It should be added before the reaction starts, not directly.

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