Student Sheet 14

Phosphatase Enzymes in Plants

Technical and Teaching Notes

Introduction

This is a standard protocol to extract phosphatase enzymes from plants, including a method
to calculate the rate of reaction. Investigations using this protocol can be found on the
Science & Plants for Schools website.

Phosphatases are enzymes widespread in plants easily extracted from germinating seeds.
The pink substrate phenolphthalein phosphate (PPP) can be used to estimate the amount of
enzymatic activity, and investigations can be carried out under differing pH and temperatures.
Students have the opportunity to use colorimeters in this practical.

Apparatus

Materials required by each student/group:

 mortar and pestle

 beaker containing 50cm3 distilled water
 filter funnel
 piece of muslin (approx. 12 cm x 12 cm)
 centrifuge tube
 test tube
 5cm3 buffer solution pH 5
 1cm3 1% phenolphthalein phosphate (PPP) solution
 beaker containing 5 cm3 10% sodium carbonate solution
 gloves and eye protection

Materials to be shared:

 water bath at 30°C

 Sodium phosphate (NaH2PO4)
 500 g packet of beansprouts (see Suppliers section)
 bench centrifuge
 colorimeter with 550 nm filter and cuvettes or test tubes as appropriate.

Science and Plants for Schools: www.saps.org.uk

Sheet 14 - Phosphatase Enzymes in Plants – Technical and Teaching Notes
Resource 292, Version 1.1 (revised 2020)
 Student Sheet 14

Preparation of materials

CARE: Wear gloves when preparing the PPP solution and the buffer.

Buffer tablets can be used. Alternatively, to make up 100 cm3 of buffer add 51.5 cm3 0.2 M
Na2HPO4 (the dibasic sodium phosphate) to 48.5 cm3 0.1 M citric acid. The pH of this mixture
should be close to 5.0. Adjust to exactly 5.0 by adding the appropriate solution drop by drop
while checking with a pH meter.

Suppliers

PPP

PPP is available from SigmaAldrich, www.sigmaaldrich.com, catalogue number P9875.

£18.30 buys enough PPP for approximately 100 assays. However, there is a standard
delivery charge of £22 no matter how small the order, so you may wish to partner with
neighbouring schools to place an order.

Care should be taken when weighing as the dust may be hazardous.

The solution should be made up just before the practical since it is unstable and gradually
degrades to free phenolphthalein which will affect the results. The solution can be stored at 4
degrees for several days without this being a problem or frozen at -20 for longer periods.

Note that if the dry powder is to be kept for more than a few weeks it should also be stored at
-20 C with a desiccant.

Phosphatase

The Phosphatase is extracted from the Mung beans.

Beansprouts

It will usually be more convenient to buy a 500 g packet of beansprouts from a supermarket.

Alternatively, soak mung bean seeds in a shallow dish and pour off excess water. Rinse two
times each day with fresh water and drain. CARE: Too much water will cause them to rot, too
little and they will dry out. Leave in a dark cupboard at about 25°C and harvest after 3 days.
When making the enzyme solution from this source 1 cm3 of water should be added for every
seedling used. Most of the water should be added after the seedlings have been crushed
using a mortar and pestle.

Science and Plants for Schools: www.saps.org.uk

Sheet 14 - Phosphatase Enzymes in Plants – Technical and Teaching Notes
Resource 292, Version 1.1 (revised 2020)
 Student Sheet 14

Background information

Phosphatase enzymes are widespread in nature and may be easily extracted from
germinating seeds.

They serve to remove phosphate groups from a wide range of organic phosphates and thus
make available a metabolic pool of phosphate ions.

The formation of ATP and nucleotides, the regeneration of ribulose bis-phosphate and the
phosphorylation of glucose at the start of the glycolytic pathway are important biochemical
processes requiring phosphate.

Some phosphatases are specific for a particular substrate (e.g. Glucose-6-phosphatase) but
others exhibit broad specificity and can cleave a phosphate group from a wide range of
organic substrates.

The basic reaction is as follows:

XPO4 ---------------------> X + PO43-

Phosphatase

The accurate estimation of phosphate is a complex process so this protocol relies on a

substrate which yields a product which is easily quantified by colorimetric methods.

Phenolphthalein phosphate (PPP) has the property of being colourless in alkaline solution,
whereas free phenolphthalein produces a pink colour. The amount of enzymatic activity can
thus be estimated by the intensity of colour produced from a standard reaction mixture when
an alkali such as sodium carbonate solution is added.

The phosphatase enzyme has an optimum pH of around 5 so the pH of the reactions should
be kept below 9.2. Any buffer system can be used, including off the shelf buffer tablets. The
practical makes use of basic laboratory apparatus, although you may need to borrow a
colorimeter from the chemists.

Since the reaction is stopped by the sodium carbonate the tubes can be stored in the
refrigerator for several days before the colour intensity is assessed. This can also be used for
reassessment of the accuracy of the technique. If duplicates are done for each variable under
investigation (which should be common practice) the colorimeter readings for each pair
should be identical.

Science and Plants for Schools: www.saps.org.uk

Sheet 14 - Phosphatase Enzymes in Plants – Technical and Teaching Notes
Resource 292, Version 1.1 (revised 2020)