Phosphorylase

Safety

Results

Methods

Potato phosphorylase can be used to demonstrate the synthesis of starch. It can also be used to investigate the equilibrium in a reversible reaction.

Phosphorylase, (1,4-α-D-glucan:phosphate α-D-glucosyltransferase, EC 2.4.1.1) is usually associated with the breakdown of starch or glygogen by the release of terminal glucose molecules as glucose-1-phosphate. However, if provided with glucose-1-phosphate and in the absence of inorganic phosphate, the reaction proceeds in the opposite direction and starch is synthesised by the addition of successive glucose molecules to pre-existing polysaccharide chains.

Starch phosphorylase extracted from potato can be used to demonstrate the synthesis reaction in the presence of glucose-1-phosphate. The reaction requires priming with small fragments of partially hydrolysed starch which can grow with the addition of further glucose molecules. It also requires the absence of inorganic phosphate since this will cause the reaction to proceed in the opposite direction.

This system can also be used as a good demonstration of reversibility and equilibrium in an enzyme reaction.

The direction of the reaction depends on the ratio of glucose-1-phosphate : inorganic phosphate. By adding phosphate during the course of the reaction the direction can be reversed and synthesis of starch becomes degradation of starch.

In both of these investigations the reaction can be followed using a colorimeter to measure the starch-iodine complex.

Synthesis of starch

The results shown below were obtained using 5cm³ 0.01M glucose-1-phosphate in 0.1M citric acid-sodium citrate buffer pH6 with 0.1cm³ primer and 0.5 cm³ of enzyme. Samples were taken by removing 0.5 cm³ into 3cm³ of iodine solution, (2.5% iodine stock solution in 0.1M HCl) The reaction temperature was 25°C. (see Methods).

Reversability of the reaction

The direction of the reaction, (i.e. synthesis or degradation of starch), depends on the ratio of glucose-1-phosphate to inorganic phosphate. The equilibrium point occurs at a phosphate:G-1-P ratio of about 7:1.
Using a phosphate-free buffer the reaction can be run in the direction of starch synthesis, then by adding a phosphate buffer it can be reversed and the starch begins to be degraded.
Comparing this with the effect of the addition of a phosphate-free buffer acts as a control, showing that in this case the reaction will continue to run in the direction of the synthesis of starch.The buffers used were 0.1M citric acid-sodium citrate buffer pH6 and 0.1M phosphate buffer pH6 (see Methods).

Extraction of potato phosphorylase

• Try to keep everything as cold as possible during this extraction.
• Use a blender, or a pestle and mortar to liquidise small pieces of potato with 0.1M citric acid-sodium citrate buffer pH6 in the ratio 2g potato : 1cm³ buffer.
• Filter the liquid through four layers of muslin and allow the filtrate to stand to let residual starch settle. Alternatively the liquid can be filtered through filter paper.

The resulting liquid can be used as it is or centrifuged to remove suspended particles.
[Further purification can be achieved by ammonium sulphate precipitation. The 35-45% saturation fraction will contain most of the activity.]

Primer

Take great care with hot acid

The synthesis of starch by potato phosphorylase can only proceed if the reaction mix is primed by the addition of small fragments of starch at least four glucose molecules in length. A satisfactory primer can easily be prepared by acid hydrolysis of soluble starch. We have found the following method gives good results.

• Place 2cm³ of a 10% solution of soluble starch in a test tube.
• In a second test tube put 1cm³ of 2M hydrochloric acid.
• Stand both test tubes in boiling water for a few minutes to heat up then mix the contents and let the mixture stand in boiling water for 4 minutes.
• Remove the test tube and add 1cm³ of 2M sodium hydroxide to stop the reactions and neutralise the pH.
Add 0.1cm³ of this primer to 5cm³ of the reaction mixture.

Reaction mixture

We have used the reaction mixture given here successfully.
• 4.4cm³ of 0.01M glucose-1-phosphate in citric acid-citrate buffer, (0.1M, pH6).
• 0.1 cm³ primer
• 0.5cm³ enzyme.

0.5cm³ samples were taken and added to 3cm³ of iodine solution containing 0.1M HCl, (iodine stock solution diluted 1:40)

The reaction can be carried out at room temperature, the results reported here were all obtained at 25°C.

Click to download a pdf file containing much of the information about this topic.