The Salivary Amylase Practical: A Conclusion That Does Not Fit the Data
In a practical, a student mixes 2 mL of 1% starch with 1 mL of salivary amylase at different temperatures and checks for starch with iodine every minute. At 37 °C the blue colour disappears in 2 minutes. At 60 °C it disappears in 9 minutes. At 0 °C the blue colour is still present at 20 minutes, but when the tube is then warmed to 37 °C it clears in a further 3 minutes. The tube kept at 100 °C for 10 minutes never clears, even after cooling to 37 °C and adding fresh starch. In a second set at 37 °C, adding 1 mL of 1% sodium chloride shortens the clearing time to 1 minute, while adding 1 mL of 1% copper sulfate stops the reaction completely. The student writes: "The enzyme works best at 37 °C because that is body temperature; therefore heat does not denature amylase, it only slows it down."
1. Use the data to identify the optimum temperature and distinguish the effects of low temperature from those of high temperature on amylase.
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Optimum: 37 °C. It gives the shortest clearing time (2 minutes), so it is the temperature of maximum observed activity under these conditions.
Low temperature (0 °C)
- Activity is very low because molecular motion and collision frequency are reduced. The enzyme is inactive, not destroyed.
- When warmed to 37 °C, the same tube clears in 3 minutes, so the enzyme has retained its structure and function. The effect is reversible inhibition by cooling.
High temperature (60 °C and 100 °C)
- At 60 °C clearing takes 9 minutes. This is slower than at 37 °C because thermal denaturation is already competing with catalysis; the enzyme is partly inactivated.
- At 100 °C there is no clearing even after cooling and adding fresh substrate. The tertiary structure has been irreversibly disrupted, so the active site is lost. This is denaturation, not reversible slowing.
Judgement on the student's conclusion: the first clause is acceptable as a description, but the second clause is contradicted by the 100 °C tube. Heat does denature amylase; the data show it directly.
2. Explain the effects of sodium chloride and copper sulfate on this reaction.
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Sodium chloride (activation)
- Chloride ions act as an activator of salivary amylase. The enzyme has a binding site for chloride that stabilises the active conformation and increases turnover.
- This is not a substrate effect: starch is unchanged, yet the reaction is faster. The ion increases the effective activity of the enzyme present.
- The result is a shorter clearing time (1 minute instead of 2 minutes) and shows that activity depends on cofactors as well as on temperature and pH.
Copper sulfate (inhibition)
- Copper(II) ions are heavy-metal ions that bind to side-chain groups such as sulfhydryl and carboxylate groups, disrupting the folded structure and blocking catalysis.
- The effect is effectively irreversible under these conditions, so adding fresh starch does not restore activity. The enzyme protein has been denatured, not merely slowed.
Contrast: chloride is a physiological activator; copper is a non-specific denaturing inhibitor. The two tubes therefore demonstrate that enzyme activity is a property of an intact protein structure plus its required ionic environment.
3. The student claims that 37 °C is optimal "because it is body temperature". Is that a valid explanation? What experiment would separate a temperature optimum from body temperature?
Read the worked answer
The explanation is not valid as stated. It is a teleological restatement, not a mechanism. The optimum arises from the balance between two opposing effects for that particular enzyme:
- Raising temperature increases kinetic energy and reaction rate.
- Raising temperature also increases the rate of unfolding and irreversible inactivation.
The observed optimum is where these balance for salivary amylase. It happens to coincide with body temperature because the enzyme functions in the mouth, but the coincidence is not the cause of the optimum.
Experiments that would distinguish the two ideas
- Pre-incubation test: hold amylase at each temperature for 10 minutes without substrate, then assay all samples at 37 °C. If activity falls after pre-incubation at high temperature, denaturation is demonstrated.
- Test another enzyme: a thermophilic enzyme with a much higher optimum would show that "body temperature" is not a general explanation.
- Measure activity over time at each temperature: a true optimum should give a reproducible peak, and the decline above it should follow loss of active enzyme.
The student's statement should be replaced by a statement about rates of catalysis versus rates of denaturation.