(Orthophosphoric-monoesterphosphohydrolase, alkaline medium 3.13.1)
(Orthophosphoric-monoesterphosphohydrolase, acid medium 184.108.40.206)
Phosphatases liberate inorganic phosphate from organic phosphate esters. Acid phosphatase (220.127.116.11) 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.
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.
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.
» 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.
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.
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