Assay of Acid Phosphatase enzyme activity from Potatoes

This is the enzymology protocol on “Assay of Acid phosphatase enzyme activity from Potatoes”. Before going to the protocol, we should know few points about phosphatase enzyme.

  • A phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group.
  • This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using energetic molecules like ATP.
  • A common phosphatase in many organisms is alkaline phosphatase.
  • Protein phosphorylation is a common post-translational modification of protein catalyzed by protein kinases, and protein phosphatases reverse the effect.

Assay of phosphatase Enzyme activity Protocol

Assay of Acid Phosphatase enzyme activity

Assay of Acid Phosphatase enzyme activity from Potatoes

Aim:

To determine the acid phosphatase enzyme activity in potatoes using Sodium-β-Glycerophosphate as Substrate

Principle:

Phosphatases liberate inorganic phosphate from organic phosphate ester. Acid phosphatase hydrolases a number of phosphomonoesters and phosphor proteins. Acid phosphatases can be assayed by measuring the amount of inorganic phosphorus released from Sodium-β-Glycerophosphate. The phosphorous released is estimated by Fiske Subbarao method.

Reagents:

  1. Citrate buffer (pH 5.6):

Preparation:

  • 1M Citric acid : Dissolve 5.25 gms of citric acid in distilled water and make up to 250ml with distilled water
  • 1M trisodium citrate: Dissolve 14.7 gms of trisodium citrate in distilled water and make up to 500ml

Mix 137ml of citric acid with 363 ml of trisodium citrate solution and set up pH 5.6 buffer solutions.

(0.02M Sodium-β-Glycerophosphate in citrate buffer.)

  1. 10% TCA (Tri Chloroacetic acid)

Enzyme Extract:

Clean and wash the potatoes with distilled water and peel out the skin. Weigh about 200gms by using rough balance. Cut it into small pieces then add 200ml of ice cold distilled water and homogenized in a grinder. Filter the homogenate using a cloth and stored the filtrate in a refrigerator.

Procedure:

Take 5ml of Buffer substrate clean and dry the test tube then preincubated for 30 minutes at 370C. At the end of the incubation period add 2.5 ml of % TCA and mix well. Keep the test tubes for another minute for room temperature and filter the contents. Take 1ml of the filtrate for the denaturation of inorganic phosphorous by Fiske-Subbarao method

Set up a control simultaneously by adding ml of TCA to 5 ml of buffer substrate followed by ml of enzyme and proceeds as per test.

To determine the protein content of the enzyme extract by biuret method. For this experiment, take 1 ml of extract adds 1 ml of water and add 3ml of biuret reagent and follows as per the biuret estimation procedure.

Report:

The amount of inorganic phosphorous present in the given unknown sample is __________ mg of inorganic phosphate formed per 1 ml of enzyme / 30 minutes.

Leave a Reply