1. Purpose requirement
Master the principles and methods of acidic phosphate (ester) enzyme activity determination.
2. Method principle
Acid phosphatase is an enzyme that exists in the body to hydrolyze organic phosphate bonds. With sodium p-nitrophenol phosphate as the substrate, under the action of acid phosphatase, hydrolyze under alkaline conditions to produce yellow p-nitrophenol, which can be measured colorimetrically with a spectrophotometer. They are ubiquitous in all kinds of seeds, and their content is relatively large. In the early stage of germination, as the germination process of seeds increases, the activity of acid phosphatase is generally proportional to the vigor of seeds.
3. Main experimental instruments and materials
Dry seeds or imbibed seeds, electronic balance, spectrophotometer, constant temperature water bath, test tube with stopper scale, small beaker, mortar, plastic tube, scissors.
4. Master points
Master the commonly used method for measuring the activity of acid phosphate (ester) enzyme-sodium nitrophenol phosphate method.
5. Experimental content
(1) Extraction of enzyme solution. Weigh 1 g of sample, grind with 5 mL of grinding buffer in an ice bath in a mortar, then rinse with 5 mL of grinding buffer, transfer to a centrifuge tube, and centrifuge at 20,000 rpm for 10 min. Aspirate the supernatant, which is the crude enzyme extract. Can be stored in the refrigerator for future use.
(2) Determination of vitality. Take 0.1-1mL of enzyme solution (depending on the amount of enzyme content), add water to 1mL, then add 1mL of buffer solution and 0.1mL of sodium p-nitrophenol phosphate solution. The blank control replaced the enzyme preparation with 1 mL of grinding buffer. After mixing well, keep it in a 30 ° C incubator for 10 minutes and shake it from time to time. After 10min, add 1m0.5mol / L NaOH solution, mix thoroughly, terminate the reaction, and make p-nitrophenol yellow. The absorbance A was measured at a wavelength of 400 nm.
(3) Calculate the enzyme activity in terms of nmol of hydrolyzed substrate per milligram of seed per minute.
Enzyme activity nmol / min.mg =
Where 0.019 is pH = 14, the μmol absorption coefficient of p-nitrophenol, that is, when the concentration of p-nitrophenol is 1mol / L, its A = 0.019;
3.1 is 0.0031 × 1000, the volume of the reaction mixture is 3.1, and μmol is converted to nmol times 1000;
V is the total volume of the enzyme preparation;
V1 is the volume of enzyme used each time.
In the experiment, the enzyme preparation should be kept at a low temperature to prevent the enzyme activity from decreasing, and it can be extracted and stored in the refrigerator.
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