Investigating the effect of pH on amylase activity
Measure the time taken for amylase to completely break down starch, by withdrawing samples at 10 second intervals and noting the time at which the solution no longer gives a blue-black colour with iodine solution (but the iodine solution remains orange). Use buffers to provide solutions at different pHs. Calculate the rate of this enzyme controlled reaction by calculating 1÷ time.
This procedure is simple enough for individuals to carry out if you have enough dimple tiles. If you choose to investigate five pHs, then groups of five students could complete the investigation by working together and pooling results.
Apparatus and Chemicals
For each group of students:
Syringes, 5 cm3, 2 (1 for starch, 1 for amylase)
Iodine solution in a dropper bottle (Note 4)
Test tube rack
Test tube, 1 for each pH to be tested
Dimple tile or white tiles
For the class – set up by technician/ teacher:
Amylase 1% (or 0.5%) (Note 1)
Starch 1% (or 0.5%) (Note 2)
Buffer solutions covering a range of pH, each with a labelled syringe/ plastic pipette (Note 3)
Health & Safety and Technical notes
Amylase solution and iodine solution are low hazard once made up. Wear eye protection when handling iodine solution.
Hazards of buffers may vary. See CLEAPSS Recipe card or supplier’s information and see Note 3.
1 Amylase (See CLEAPSS Hazcard and Recipe card) The powdered enzyme is HARMFUL, but solutions less than 1% are LOW HAZARD. It is wise to test, well in advance, the activity of the stored enzyme at its usual working concentration to check that substrates are broken down at an appropriate rate. Enzymes may degrade in storage and this allows time to adjust concentrations or to obtain fresh stocks. Amylase will slowly lose activity, so it is best to make up a fresh batch for each lesson; batches may vary in activity and results collected on different days will not be comparable. The optimum temperature for your enzyme will be listed on the supplier’s label.
Using saliva: the CLEAPSS Laboratory Handbook provides guidance on precautions to take (including hygiene precautions) in order to use saliva safely as a source of amylase. This has the advantage of being cheaper, not requiring technicians to make up fresh solutions each lesson, it is directly interesting to students, and salivary amylase is reliable. It also provides an opportunity to teach good hygiene precautions – including ensuring that students use only their own saliva samples (provide small beakers to spit into); that students are responsible for rinsing their own equipment; and that all contaminated glassware is placed in a bowl or bucket of sodium chlorate(I) before technicians wash up.
2 Starch suspension – make fresh. Make a cream of 5 g soluble starch in cold water. Pour into 500 cm3 of boiling water and stir well. Boil until you have a clear solution. Do not use modified starch.
3 Buffers: (See CLEAPSS Recipe card) If you make universal buffer it will contain sodium hydroxide at approximately 0.25 M, and should be labelled IRRITANT. Refer to other relevant Hazcards if you choose to make other buffers, or to supplier’s information if you purchase buffer solutions/ tablets. (Note 1)
4 Iodine solution (See CLEAPSS Hazcard and Recipe card). A 0.01 M solution is suitable for starch testing. Make this by 10-fold dilution of 0.1 M solution. Once made, the solution is a low hazard but may stain skin or clothing if spilled.
There are no ethical issues associated with this procedure.
SAFETY: All solutions once made up are low hazard. Wear eye protection, as iodine may irritate eyes.
a Check the speed of the reaction with the suggested volumes of reactants to be used – 2 cm3 of starch: 2 cm3 of amylase: 1 cm3 of buffer at pH 6. Ideally the reaction should take about 60 seconds at this pH: this is the usual optimum for amylase (see note 1). If the reaction is too fast, either reduce the enzyme volume or increase the starch volume. If the reaction is too slow, increase the enzyme volume or concentration or reduce the starch volume or concentration.
b Place single drops of iodine solution in rows on the tile.
c Label a test tube with the pH to be tested.
d Use the syringe to place 2 cm3 of amylase into the test tube.
e Add 1 cm3 of buffer solution to the test tube using a syringe.
f Use another syringe to add 2 cm3 of starch to the amylase/ buffer solution, start the stop clock and leave it on throughout the test. Mix using a plastic pipette.
g After 10 seconds, use the plastic pipette to place one drop of the mixture on the first drop of iodine. The iodine solution should turn blue-black. If the iodine solution remains orange the reaction is going too fast and the starch has already been broken down. Squirt the rest of the solution in the pipette back into the test tube.
h Wait another 10 seconds. Then remove a second drop of the mixture to add to the next drop of iodine.
i Repeat step h until the iodine solution and the amylase/ buffer/ starch mixture remain orange.
j You could prepare a control drop for comparison with the test drops. What should this contain?
k Count how many iodine drops you have used, each one equalling 10 seconds of reaction time.
l Repeat the whole procedure with another of the pH buffers to be used, or pool the class results.
m Consider collecting repeat data if there is time.
n Plot a graph of time taken to break down starch against pH, or calculate the rate of reaction and plot rate against pH.
This is a straightforward practical giving reliable, unambiguous results. The main errors will be in the order of mixing the enzyme/ substrate/ buffer, or a delay in sampling so that the reaction time is under-estimated or rate is over-estimated. Temperature variation affects enzyme activity, so results collected on different days are not comparable.
Health and safety checked, September 2008
Download the student sheet Investigating the effect of pH on amylase activity (72 KB) with questions and answers.
Royal Society of Chemistry: Chemistry for Biologists: Enzymes
A clear and thorough presentation of information about enzymes as chemical catalysts and the factors affecting their activity.
(Website accessed October 2011)