Determination of sucrase activity in plant tissues

Summary

To understand the method of extracting sucrase from plant tissues and the principle of Nelson's method for determining sucrase activity.

Principle

The basic principle for the determination of sucrase activity in plant tissues is that sucrase hydrolyzes non-reducing sucrose to glucose and fructose, and glucose, as a reducing sugar, contains free aldehyde groups, which reduce ^Cu2+ in alkaline solution, and the reducing sugar is oxidized to hydroxy acid. The activity of sucrase can be determined by the formation of a blue complex between the reagent and cuprous oxide, and the absorption peak at 510 nm is directly proportional to the concentration of reducing sugar, and the range of the method is 25-200 昭.

Operation method

Determination of sucrase activity in plant tissues

Principle

The basic principle for the determination of sucrase activity in plant tissues is that sucrase hydrolyzes non-reducing sucrose to glucose and fructose, and glucose, as a reducing sugar, contains free aldehyde groups, which reduce Cu2+ in alkaline solution, and the reducing sugar is oxidized to hydroxy acid. The activity of sucrase can be determined by the formation of a blue complex between the reagent and cuprous oxide, and the absorption peak at 510 nm is directly proportional to the concentration of reducing sugar, and the range of the method is 25-200 昭.

Materials and Instruments

Material: Plant leaves.
Equipment: spectrophotometer, graduated stoppered test tubes, thermostatic water bath, pipettes.
Reagents:
①4 mmol - L
- 1
Glucose solution 20 mL
②4 mmol - L
①4 mmol - L 1 glucose solution 20 mL
Sucrose solution 20 mL
③0.5 mmol - L
L-1 sucrose solution 200 mL
Sucrose solution 200 mL
④0.2 mol-L-1 sucrose solution 200 mL
④0. 2 mol - L -1
Acetic acid buffer (pH 4.5) 200 mL
Nelson's reagent:
⑤ A Reagent: 100 mL of solvent containing Na
⑤A Reagent: 100 mL of solvent containing Na
CO
⑤A Reagent: 100 mL of solvent containing Na 2 CO
2.5 g,NaHCO
2.5 g, NaHCO
2.0 g,Na
2
SO
4
20 g, 20 g of sodium potassium tartrate.
⑥B Reagent: 100 mL of solvent containing CuSO
.
- 5 H
2
O 15 g, concentrated H
2
SO
4
2 drops. Mix with the ratio of A:B=50:2 to use, and dissolve above 37 °C before use to prevent precipitation of solutes.
(7) Reagent for pincer acid: 100 mL contains 5 g of key acid, concentrated H
2
SO
4. 2 mL, monumental acid reagent
4.2 mL, 0.6 g of sodium stelarate.

Move

The basic procedure for the determination of sucrase activity in plant tissues can be divided into the following steps:1 . Standard curve preparation：

（1） Take 9 stoppered test tubes and add samples according to Table 49-1.Table 49-1 Sampling table for standard curve preparation mL

Reagent				Tube number
	1	2	3	4	5	6	7	8
4 mmol - L-1 glucose solution	0.02% 0.02% 0.02% 0.02% 0.02% 0.02	0.02	0.02 0.05	0.05 0.1	0.15	0.2	0.25	0.3
Water	1.0	0.98	0.95	0.9	0.85	0.8	0.75	0.7
OD5io nm

(2) Add 1 mL of Nelson's reagent to each tube, cover with a stopper, and place in a boiling water bath for 20 min. cool to room temperature, and add 1 mL of monumental key acid reagent to each tube.(3) After 5 min, add 7 mL of vaporized water to each tube and mix well.(4) Measure the optical density at 510 nmT, and make a standard curve with reducing sugar glucose as the horizontal coordinate and ODs as the vertical coordinate.(2) Determination of enzyme activity: Take 2 g of wheat seedling, add 2 mL of acetic acid buffer, grind it into a paste with a mortar and pestle in an ice bath, and centrifuge it at 12 000 r-min 1 for 10 min, and leave the supernatant for the determination of enzyme activity. Add 0.8 mL of acetic acid buffer, 0.2 mL of 0.5 mmol-sucrose solution, and 1 mL of diluted enzyme solution to each of 2 stoppered graduated test tubes, and use the same treatment without enzyme solution as the blank control. After cooling to room temperature, add 1 mL of Nelson's reagent to each tube, and after 5 min, add 7 mL of distilled water to each tube, and measure the optical density OD at 510 nmT.3. Calculation of results: At room temperature and pH 4.5, the amount of enzyme required to hydrolyze 1 day of mol glucose per minute is defined as 1 unit of enzyme activity (U).Sucrase enzyme activity (U・g- 1 .) T, Measured reducing sugar content XnXVXl 0005 ) - WXt where: 〃 - dilution factor;V - total volume of enzyme solution, mL;W a sample weight, g;W a sample weight, g; t a time, 10 min;1 000-millimolar converted to micromolar multiples.

Caveat

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

2. glucose should be dried to constant weight at 80°C in a constant temperature oven when preparing glucose standards.

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