Investigating Enzyme Activity: Catalase and Amylase

Enzymes are invisible, but their effects are easy to measure. This guide covers two classic school practicals: catalase, which breaks down hydrogen peroxide into oxygen gas that you can collect and measure, and amylase, which digests starch, tracked with iodine solution. Both let you investigate how temperature, pH or concentration affect enzyme activity, and both give real data you can graph and analyse.

For the theory behind the results, see how temperature and pH affect enzymes.

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Practical 1: catalase and hydrogen peroxide

Purpose

To measure the rate at which catalase breaks down hydrogen peroxide, and to investigate how one factor (for example, substrate concentration or temperature) affects that rate.

Principle

Hydrogen peroxide is a toxic by-product of metabolism. The enzyme catalase, found in almost all living cells, breaks it down:

2H₂O₂(aq) → 2H₂O(l) + O₂(g)

The oxygen can be collected and measured. The faster the gas is produced, the higher the enzyme activity. Potato, liver, celery and yeast are all good sources of catalase.

Equipment

• Potato (or yeast suspension)
• Cork borer and white tile, scalpel, ruler (to cut equal-sized potato discs)
• Hydrogen peroxide solutions, no stronger than about 3% (10 volume), diluted to a range such as 0.5%, 1%, 1.5%, 2%, 2.5% and 3%
• Conical flask or boiling tube with a bung and delivery tube
• Upturned measuring cylinder in a trough of water, or a gas syringe
• Stopwatch
• Thermometer and water baths (for the temperature version)
• Eye protection

Safety

• Wear eye protection. Hydrogen peroxide is an irritant and can damage eyes; stronger solutions are corrosive. Keep concentrations at 3% or below.
• Take care with scalpels and cork borers; cut on a tile, away from your hand.
• Hydrogen peroxide can bleach skin and clothes; wash off splashes.
• Follow your school’s risk assessment (see risk assessment in chemistry).

Method (varying substrate concentration)

1. Cut potato discs of equal size and thickness, for example 10 discs each 1 mm thick from a single cylinder. Using identical discs keeps the amount of enzyme constant.
2. Fill the measuring cylinder with water and invert it in the trough, or set up the gas syringe.
3. Put 10 cm³ of 0.5% hydrogen peroxide into the flask.
4. Add the 10 potato discs, immediately insert the bung and start the stopwatch.
5. Record the volume of oxygen collected every 30 seconds for 3 minutes.
6. Repeat with each concentration, using fresh discs each time.
7. Repeat the whole experiment at least twice more to check repeatability.

Variables:

• Independent: hydrogen peroxide concentration.
• Dependent: volume of oxygen produced per unit time.
• Controlled: temperature, volume of peroxide, number and size of potato discs, source of potato, time of measurement.

(See independent, dependent and control variables for more on planning fair tests.)

Example results

H₂O₂ concentration (%)	O₂ collected in 1 minute (cm³), mean of 3
0.5	4.0
1.0	7.5
1.5	10.5
2.0	12.5
2.5	13.5
3.0	14.0

Analysis

• Plot volume of oxygen against time for each concentration. The initial gradient (the steepest part at the start) gives the initial rate in cm³ per second or per minute.
• Plot initial rate against concentration. The rate rises steeply at first, then levels off. At high concentrations, the catalase is saturated: all active sites are busy, so extra substrate has little effect (see enzyme kinetics).

Sources of error

• Gas escapes before the bung is inserted. Practise the transfer, or add the discs through a side tube.
• Uneven disc sizes change the amount of enzyme.
• Reading the measuring cylinder under water is imprecise; a gas syringe is more accurate.
• Oxygen dissolves slightly in water, so some isn’t collected.
• Temperature drift during the experiment, since the reaction releases a little heat.

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Practical 2: amylase and starch

Purpose

To investigate how temperature (or pH) affects the rate at which amylase breaks down starch.

Principle

Amylase hydrolyses starch into maltose. Iodine solution turns blue-black with starch but stays orange-brown when no starch is left (see the iodine test for starch). By testing drops of the reaction mixture at regular intervals, you can find the time taken for all the starch to be digested. The shorter the time, the faster the enzyme.

Equipment

• Amylase solution (about 1%)
• Starch suspension (about 1%)
• Iodine solution in a dropper bottle
• Spotting tile
• Water baths at several temperatures (e.g. 20, 30, 40, 50 and 60 °C) or buffer solutions of different pH
• Test tubes, syringes or graduated pipettes, dropping pipettes
• Stopwatch, thermometer
• Eye protection

Safety

• Eye protection throughout. Iodine solution stains and irritates.
• Amylase can cause allergic reactions if inhaled as powder; use ready-made solutions and avoid creating dust.
• Take care with hot water baths.

Method (varying temperature)

1. Put one drop of iodine solution into each dimple of the spotting tile.
2. Place 2 cm³ of amylase and 2 cm³ of starch in separate test tubes in the water bath for 5 minutes so both reach the bath temperature.
3. (If investigating pH, add 1 cm³ of buffer to the starch tube.)
4. Pour the amylase into the starch, mix, and start the stopwatch.
5. Every 30 seconds, transfer one drop of the mixture to the next dimple of iodine.
6. Stop when the iodine stays orange-brown. Record the time.
7. Repeat at each temperature, and repeat each temperature three times.

Example results

Temperature (°C)	Time for starch to disappear (s), mean	Rate = 1000 ⁄ time (s⁻¹)
20	300	3.3
30	180	5.6
40	90	11.1
50	150	6.7
60	Starch still present at 600	~0

Analysis

• Calculating 1000 ⁄ time gives a quantity proportional to the rate (the factor 1000 just makes the numbers easier to read).
• Plotting rate against temperature shows the typical curve: rising to an optimum near 40 °C, then falling as the amylase denatures at higher temperatures.
• The optimum found this way is only approximate. Testing more temperatures near the peak, such as 35, 40 and 45 °C, would locate it more precisely.

Sources of error

• Sampling interval: testing only every 30 seconds means the true end point could be up to 30 seconds earlier. Shorter intervals improve precision.
• Judging the end point: the change from blue-black through purple to orange-brown is gradual and subjective. Agree on a colour standard, or use a colorimeter.
• Temperature control: tubes cool when removed from the bath; keep the mixture in the bath between samples.
• Carry-over: a dirty dropper can transfer starch between samples.

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Extending the investigations

• Enzyme concentration: use different numbers of potato discs, or different dilutions of amylase.
• pH: use buffer solutions (for example pH 4, 5, 6, 7 and 8) with amylase.
• Inhibitors: add a small amount of copper(II) sulfate solution to show how heavy-metal ions reduce catalase activity (see competitive vs non-competitive inhibition).
• Boiled controls: repeat with boiled potato or boiled amylase to show that the activity comes from the enzyme; boiled enzyme gives little or no reaction.

Writing it up

A good write-up includes a clear hypothesis, a list of variables, a results table with units and means, a graph with a line of best fit, an explanation using collision theory and active sites, and an evaluation of errors. See how to write a lab report and a lab report example.

Key takeaways

• Catalase activity can be measured from the volume of oxygen produced from hydrogen peroxide; rate rises with substrate concentration until the enzyme is saturated.
• Amylase activity can be measured by timing how long starch takes to disappear, tested with iodine.
• Rate = 1 ⁄ time (or 1000 ⁄ time) turns end-point times into rates for graphing.
• Keep the amount of enzyme, volumes and temperature controlled, repeat readings, and use boiled enzyme as a control.
• The results illustrate the ideas in enzymes: how they work.