Amylase, pH and a Flawed Iodine Assay
Amylase breaks down starch. A student mixed 2 cm3 of 1% starch with 1 cm3 of pH buffer and 1 cm3 of 0.5% amylase at 25°C, then removed samples every 30 s and tested with iodine. Times to reach a colour that gave no blue-black with iodine were: pH 3, 240 s; pH 5, 90 s; pH 7, 20 s; pH 9, 45 s; pH 11, still blue-black at 300 s. For pH 3 only, the student added amylase before the buffer and started timing 20 s later. The 25°C water bath was actually 35°C for the pH 9 tube because a second bath was used. The student concludes that pH 11 denatures amylase completely and that pH 7 is the optimum.
1. Identify the optimum pH from the data and evaluate the conclusion that pH 11 denatures amylase completely.
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The shortest time is at pH 7 (20 s), so pH 7 is the optimum among the tested values. The pH 11 result only shows that no starch breakdown was detected by the iodine test within 300 s. It does not prove complete denaturation: the enzyme might be reversibly inhibited, or the reaction might be extremely slow. Also, the iodine test is less reliable at pH 11 because iodine reacts with alkali. To test denaturation, incubate the enzyme at pH 11, then return it to pH 7 and assay activity; if activity does not return, denaturation is supported. A control without enzyme at pH 11 is needed to show that the buffer alone does not affect the iodine colour.
2. Evaluate the two procedural flaws: adding amylase before the buffer at pH 3, and using a 35°C bath for pH 9. How do they affect the conclusion?
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Adding amylase before the buffer at pH 3 means the enzyme was initially near neutral pH and could hydrolyse some starch before the acid was added. This would shorten the measured time, so the true pH 3 time is likely longer than 240 s, making the pH 7 optimum stronger. The pH 9 tube at 35°C instead of 25°C would react faster, so 45 s underestimates the time at 25°C. Correcting this would make pH 9 slower and again strengthen pH 7 as the optimum. Both flaws affect the shape of the curve, but not the main conclusion.
3. Describe a control and a quantitative method that would make the investigation more reliable.
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Use a boiled-enzyme control at each pH: boil the amylase before adding it to starch and buffer, then test with iodine. If the starch still disappears, something other than active enzyme is breaking it down or the iodine test is failing. Also run a buffer-only control to check that the pH buffer does not change the iodine colour. To measure rate rather than a single end-point, take samples at fixed intervals and use a colorimeter to measure the absorbance of the starch–iodine complex; the initial slope gives a quantitative rate. Keep all tubes in the same water bath and add the buffer before the enzyme in every trial.