Methodology for Enzymes

(Orthophosphoric-monoesterphosphohydrolase, alkaline medium 3.13.1)
(Orthophosphoric-monoesterphosphohydrolase, acid medium

Phosphatases liberate inorganic phosphate from organic phosphate esters. Acid phosphatase ( hydrolyzes a number of phosphomonoesters and phosphoproteins. Alkaline phosphatase catalyses the hydrolysis of numerous phosphate esters, such as esters of primary and secondary alcohols, sugar alcohols, phenols and amines. Phosphodiesters are not hydrolyzed by either of them.

The enzyme phosphatase hydrolyzes p-nitrophenol phosphate. The released p-nitrophenol is yellow in color in alkaline medium and is measured at 405nm. The optimum pH for acid and alkaline phosphatases are 5.3 and 10.5 respectively.

i) Acid Phosphatase
ii) Assay Using Glycerophosphate

(i) Acid Phosphatase

» Sodium Hydroxide 0.085N
   Dissolve 0.85g sodium hydroxide in 250mL water.
» Substrate Solution
   Dissolve 1.49g EDTA, 0.84g citric acid and 0.03g p-nitrophenyl phosphate in 100mL water and adjust to pH 5.3.
» Standard
   Weigh 69.75mg p-nitrophenol and dissolve in 5.0mL distilled water (100mM).
» Enzyme Extract
   Homogenize 1g fresh tissue in 10mL of ice-cold 50mM citrate buffer (pH 5.3) in a pre-chilled pestle and mortar. Filter through four layers of cheese cloth. Centrifuge the filtrate at 10,000g for 10 min. Use the supernatant as enzyme source.

Incubate 3mL of substrate solution at 37°C for 5 min.
Add 0.5mL enzyme extract and mix well.
Remove immediately 0.05mL and mix it with 9.5mL of sodium hydroxide 0.085N. This corresponds to zero time assay (blank).
Incubate the remaining solution (substrate + enzyme) for 15min at 37°C.
Draw 0.5mL sample and mix it with 9.5mL sodium hydroxide solution.
Measure the absorbance of blank and incubated tubes at 405nm.
Take 0.2 to 1.0mL (4 to 20mM) of the standard, dilute to 10.0mL with NaOH solution. Read the color and draw the standard curve.

Specific activity is expressed as m moles p-nitrophenol released per min per mg protein.

For alkaline phosphatase extract the enzyme in 50mM glycine NaOH buffer pH 10.4.
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 375mg glycine, 10mg magnesium chloride, 165mg p-nitrophenyl phosphate in 42mL of 0.1N sodium hydroxide and dilute to 100mL. Adjust to pH 10.5.

(ii) Assay Using Glycerophosphate
Acid and alkaline phosphatases can also be assayed by measuring the amount of inorganic phosphorus released from sodium glycerophosphate. Phosphorus is estimated by Fiske-Subbarow method as described below.

» Substrate: 0.1M solution of /3-glycerophosphate 3.153g in 100mL water.
» 10%TCA
» Ammonium Molybdate (2.5%)
   Dissolve 25g ammonium molybdate in 400mL of water. Add 500mL of 10 N H2SO4 and make up the volume to one liter with water.
» 1-Amino 2-Naphthol 4-Sulphonic Acid (ANSA) Reagent
   Dissolve 30g sodium metabisulphite, 6g sodium sulphite and 5.00mg ANSA separately in small quantities of water. Combine all the solutions and make up to 250mL with water. Allow to stand overnight and filter. Store refrigerated in an amber-colored bottle. Prepare fresh reagent every fortnight.
» Standard Phosphate Solution
   Dissolve 439mg potassium dihydrogen phosphate in water, add 10mL of 10N H2SO4 and make up to one liter with water (1mL = 0.1mg P).
      Add 0.5mL chloroform as preservative.
      Dilute 10mL of the above stock solution to 50mL with water and use as working standard (1mL = 20mg P)
» 0.2M solution of magnesium acetate -4.289g in 100mL water.

Pipette out 2mL of respective (acid or alkaline) buffer in a test tube.
Add 0.5mL magnesium acetate solution.
Add 2mL of b-glycerophosphate solution.
Make up the volume to 7mL with the buffer.
Add 1mL enzyme extract and incubate for 1h at 37°C.
Stop the reaction by the addition of 2mL of 10% TCA.
For the control add the enzyme after incubation.
Centrifuge at 10,000g for 10min.
Pipette out different known aliquots of supernatant of test, and control into test tubes. Make up the volume in each tube to 5mL by adding water.
10 Add 1mL of ammonium molybdate solution to each tube and mix thoroughly.
Add 0.4mL of ANSA reagent to each tube and mix well. After standing for 10min dilute to 10mL. Read the intensity of blue color at 660nm against a reagent blank.
Pipette out different aliquots of phosphate solution (5 — 50mg P) into test tubes and treat as described above.   A reagent blank is also prepared with water in place of phosphate solution. Plot absorbance versus phosphorus concentration. Read the phosphorus content from the calibration curve and express enzyme activity as the amount of phosphorus released in unit time per mg protein.

1.  Any traces of phosphate impurity in the distilled water interferes with the determination. This is easily noticed from the reagent blank which should actually be colorless.
2.  After developing, the blue color intensifies with the progress of time. It is preferable to read the tubes 10min after adding ANSA reagent.

1.  Lowry, O H, Roberts, N R, Mei-Ring, W U, Hixon, W S and Crawford, E J (1954) JBiol Chem207 19.
2.  Fiske, C H and Subba Row, Y (1925) JBiol Chem 66 575.
3.  King, EJ (1932) Biochem J 26 292.