How do you do the enzymes required practical?

What does the enzymes practical investigate?

It tests how pH affects the rate at which the enzyme amylase breaks down starch into sugar. At each pH you time how long it takes for all the starch to go, checking for starch with iodine solution as you go. Iodine turns blue-black while starch is left, and stays orange-brown once it has gone.

It is one of AQA's required practicals in both GCSE Biology and Combined Science: Trilogy, in the organisation topic. It shows the idea of an optimum pH: each enzyme works fastest at one pH and more slowly either side of it.

What equipment do you need?

Besides amylase and starch solutions, you need a buffer solution for each pH you test and iodine solution to check for starch. A water bath keeps the temperature steady.

• Amylase solution
• Starch solution
• Buffer solutions at a range of pH values, for example pH 4, 5, 6, 7 and 8
• Iodine solution in a dropping bottle
• A spotting tile
• A water bath, or a beaker of water with a thermometer
• Test tubes and a rack
• Syringes or measuring cylinders, to measure volumes
• Dropping pipettes
• A stopclock
• Eye protection

What is the method?

Bring the amylase and starch to the same temperature, mix them with a buffer, then test a drop of the mixture with iodine every 30 seconds until no starch is left. Repeat for each pH.

• Put a drop of iodine solution into each well of a spotting tile.
• Set the water bath to a fixed temperature, such as 35 °C.
• Measure a set volume of amylase solution into a test tube, and add a set volume of one buffer solution.
• Measure a set volume of starch solution into a second tube. Stand both tubes in the water bath for about 5 minutes, so they reach the same temperature.
• Pour the starch into the amylase and buffer, mix, and start the stopclock straight away.
• Keep the tube in the water bath. Every 30 seconds, use a pipette to put one drop of the mixture into the next well of iodine.
• When a drop leaves the iodine orange-brown, all the starch has been broken down. Record that time.
• Repeat with each of the other buffers, then repeat the whole set so you can calculate a mean for each pH.

What are the variables?

The pH is the one thing you change. Temperature is the control variable to watch most closely, because enzymes are sensitive to it too.

How do you work out and present the results?

Find the mean time at each pH, leaving out anything anomalous, then turn each time into a rate. A shorter time means a faster reaction, so rate = 1 ÷ time, with the unit s⁻¹ (per second). Using 1000 ÷ time gives the same pattern with numbers that are easier to plot.

Worked example: at pH 6, three repeats take 90, 120 and 90 seconds. The mean is 300 ÷ 3 = 100 seconds, so the rate is 1000 ÷ 100 = 10. The repeats range from 90 to 120 seconds. At pH 8 the mean is 250 seconds, giving a rate of 1000 ÷ 250 = 4.

Plot rate on the y-axis against pH on the x-axis and draw a smooth curve of best fit. It should rise to a peak and fall on either side. The peak is the optimum pH for your amylase. Away from the optimum, the shape of the enzyme's active site changes, so starch fits less well and the reaction slows. At extreme pH values the enzyme is denatured and the reaction stops.

How do you make it accurate and safe?

Your time can only be as precise as your sampling. With a drop every 30 seconds, the starch really ran out at some point between the last blue-black drop and the first orange-brown one, and sampling every 10 seconds narrows that gap. Let both tubes reach the water bath temperature before you mix them, and start the clock the moment they meet. Decide on a cut-off time before you start, such as 10 minutes, in case the starch never fully goes at one of the pH values.

Iodine solution can irritate your eyes and stains skin, so wear eye protection. Enzymes such as amylase can cause allergic reactions, so wash off any splashes on your skin. Take care with hot water.

What do exam questions ask about it?

Expect to be asked why a buffer is used: it keeps the pH fixed at the value being tested. Another common question is why the tubes stand in the water bath before they are mixed. Calculations use rate = 1000 ÷ time or 1 ÷ time, and you may read the optimum pH off a graph. An anomaly might come from a mixture that started reacting before it reached temperature. A common improvement is to test more pH values close to the peak, to find the optimum more precisely. The 6-mark version asks you to describe the method.

An exam-style question we wrote: "At pH 5, the starch was fully broken down after 150 seconds. Calculate the rate of reaction using rate = 1000 ÷ time. Give your answer to 2 significant figures." 1000 ÷ 150 = 6.666..., which is 6.7 to 2 significant figures.

Common mistakes

• Saying the enzyme is "killed". Enzymes are not alive: at an extreme pH they are denatured.
• Mixing the solutions before they reach the water bath temperature, so the reaction starts at a different temperature.
• Treating a long time as a fast reaction. The longer the starch takes to go, the slower the rate.
• Writing that the active site "changes" without saying why it matters: the starch no longer fits.