In this article we will discuss about the tests for estimation of acid phosphatase in plants.
Principle:
The enzyme phosphatase hydrolyzes p-nitro-phenol phosphate. The released p-nitro-phenol is yellow in colour in alkaline medium and is measured at 405 nm. The optimum pH for acid and alkaline phosphatases are 5.3 and 10.5, respectively.
Materials:
(i) Sodium Hydroxide 0.085 N:
Dissolve 0.85 g sodium hydroxide in 250 ml water.
(ii) Substrate Solution:
Dissolve 1.49 g EDTA, 0.84 g citric acid and 0.03 g p-nitro-phenyl phosphate in 100 ml water and adjust to pH 5.3.
(iii) Standard:
Weigh 69.75 mg p-nitro-phenol and dissolve in 5.0 ml distilled water (100 mM).
(iv) Enzyme Extract:
Homogenize 1 g fresh tissue in 10 ml of ice-cold 50 mM citrate buffer (pH 5.3) in a pre-chilled pestle and mortar. Filter through four layers of cheese cloth. Centrifuge the filtrate at 10,000 g for 10 min. Use the supernatant as enzyme source.
Test Procedure:
1. 3 ml of substrate solution is incubated at 37°C for 5 min.
2. 0.5 ml enzyme extract is added and mixed well.
3. Immediately 0.05 ml is removed and mixed it with 9.5 ml of sodium hydroxide 0.085 N. This corresponds to zero time assay (blank).
4. The remaining solution (substrate + enzyme) is incubated for 15 min at 37°C.
5. 0.5 ml sample is drawn and mixed with 9.5 ml sodium hydroxide solution.
6. The absorbance of blank and incubated tubes is measured at 405 nm.
7. 0.2 to 1.0 ml (4 to 20 mM) of the standard is taken, and diluted to 10.0 ml with NaOH solution.
The colour is read. Then draw the standard curve.
Calculation:
Specific activity is expressed as m moles p-nitro-phenol released per min per mg protein.
Notes:
1. For alkaline phosphatase extract the enzyme in 50 mM glycine NaOH buffer pH 10.4.
2. Alkaline phosphatase functions optimally at about pH 10.5. The assay procedure is similar to that for acid phosphatase, except for the substrate solution.
Prepare the substrate solution as follows:
Dissolve 375 mg glycine, 10 mg magnesium chloride, 165 mg p-nitro-phenyl phosphate in 42 ml of 0.1 N sodium hydroxide and dilute to 100 ml. Adjust to pH 10.5.