Salivary Amylase: Separating pH, Temperature and Iodine Colour
During a practical on enzyme activity, students use salivary amylase to digest 1% starch. They set up tubes at pH 3, pH 7, and pH 9 using buffer, and at 20 °C, 37 °C, and 60 °C. After 10 minutes, each tube is tested with iodine. The pH 7/37 °C tube gives a pale yellow colour; the pH 3 and 60 °C tubes remain blue-black; the pH 9 tube is purple. A student concludes that pH 3 and 60 °C both 'destroy' the enzyme in the same way and that pH 9 is the optimum. The teacher points out that the buffers were added before the enzyme and that the iodine test only shows remaining starch, not enzyme activity. The class must separate the effects of pH and temperature and decide what can be concluded from the colours alone.
1. Explain why the blue-black result at pH 3 does not by itself prove that the enzyme is denatured. Describe an experiment that would distinguish denaturation from reversible inhibition.
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A blue-black iodine result means starch remains; it does not show why. Low pH can change the ionisation of amino-acid side chains in the active site, reduce substrate binding, or cause partial unfolding. Some of these effects are reversible if the enzyme is returned to pH 7; true denaturation is not. To distinguish them, pre-incubate amylase at pH 3 for 10 minutes, then rapidly adjust a sample to pH 7 and add starch; if starch is digested, the low pH was inhibitory/reversible. A parallel sample kept at pH 3 serves as control. For heat, pre-incubate at 60 °C, cool to 37 °C, then assay; no recovery supports denaturation. Include a time course because iodine at 10 minutes cannot distinguish slow activity from no activity.
2. The pH 9 tube is purple, not blue-black. What does this indicate, and why is it wrong to call pH 9 optimum from this single result?
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Purple is an intermediate iodine colour: some starch has been digested, but not all. That means the enzyme is active at pH 9, though less effective than at pH 7 under these conditions (where the result is pale yellow). It is wrong to call pH 9 optimum because 'optimum' requires the highest rate, not merely detectable activity. The comparison is also uncontrolled: only one time point, one starch concentration, and one enzyme amount were used. A proper determination measures the initial rate over a pH range (e.g. 5–9) with fixed substrate and enzyme, and plots rate against pH. The peak of that curve is the optimum. The purple tube alone cannot distinguish a lower Vmax from a slower approach to completion.
3. Identify one controlled variable the students failed to control and explain how it could change the conclusion.
Read the worked answer
A key uncontrolled variable is the time and temperature at which each tube actually incubated, or the volume/activity of saliva added. If the 60 °C tube was placed in the bath before it reached temperature, or if saliva was added before the buffer equilibrated, the enzyme experienced a different temperature history. Likewise, if the pH 3 tube received enzyme last, its effective incubation was shorter. Either error can make a tube look inactive and be misread as denaturation. Control by pre-equilibrating buffer and starch at the target temperature, adding a fixed volume of freshly diluted enzyme at time zero, and stopping all tubes at the same elapsed time. Repeats and a no-enzyme blank are also needed.