Core practical: pH and amylase

Amylase is a biological catalyst: it speeds up a reaction without being used up. Its substrate binds at a complementary active site. A pH change can make binding less successful by changing the active site, so controlling other conditions matters.

The investigation asks: How does pH affect the rate at which amylase breaks down starch? Amylase is the enzyme, starch is its substrate, and a buffer provides and maintains a chosen pH. Iodine solution is used to monitor whether starch is still present.

Iodine is orange-brown when no starch is detected and turns blue-black when starch is present. Therefore, a blue-black sample is an observation; from it, you infer that starch remains. The endpoint is the first sample that leaves the iodine orange-brown, supporting the inference that the starch is no longer detected.

Apparatus and arrangement

Use dilute prepared amylase solution, starch solution, buffer solutions covering a suitable pH range, iodine solution, test tubes and rack, separate labelled syringes, a dropping pipette, a white spotting tile, thermometer and stop clock. For the specified core practical, use a beaker of water warmed with a Bunsen burner as the water bath, with a tripod, gauze and heatproof mat. Monitor it with a thermometer and control the heating under teacher supervision so the reaction temperature stays constant. A thermostatically controlled bath is a useful alternative when evaluating temperature control, but you should also understand the specified beaker-and-Bunsen arrangement.

The solutions are measured with syringes because the volumes are small and must be kept the same. A separate syringe for each solution prevents contamination that could change pH or start starch breakdown early.

Variables

• Independent variable: pH of the buffer, for example pH 4, 5, 6, 7, 8 and 9.
• Dependent variable: time in seconds until starch is no longer detected; process this as relative rate in $\mathrm{s}^{-1}$.
• Controlled variables: temperature; volumes and concentrations of amylase and starch; volume of buffer; sampling interval; drop size; mixing method; and the batches of solutions used.

Reproducible method

1. Put one equal-sized drop of iodine into each well along a row of a white spotting tile. Prepare a reference well containing iodine plus unreacted starch, which should be blue-black, and a reference well of iodine with water, which should remain orange-brown.
2. Warm the beaker water bath gently with the Bunsen burner to a chosen temperature, such as $35\,^{\circ}\mathrm{C}$. Monitor it with a thermometer throughout. Turn off the burner when the bath reaches the chosen temperature, and adjust the heating under supervision as needed to keep the bath at that temperature rather than allowing it to keep warming.
3. Label a test tube with the first pH. Add $2.0\,\mathrm{cm}^{3}$ amylase and $1.0\,\mathrm{cm}^{3}$ of that buffer. Put $2.0\,\mathrm{cm}^{3}$ starch in a second labelled tube. Leave both in the water bath long enough to reach the set temperature.
4. Add the starch to the amylase-buffer mixture, mix in a standardised way, and start the stop clock immediately. Adding starch starts the reaction.
5. After 10 seconds, use the dropping pipette to transfer one drop of reaction mixture to the first iodine well. Do not let the pipette tip touch the iodine.
6. Sample a fresh drop every 10 seconds into successive wells. Record the first time at which the iodine remains orange-brown. If the 30-second sample is blue-black and the 40-second sample is orange-brown, the endpoint occurred between 30 and 40 seconds and is recorded by this method as 40 seconds.
7. Repeat the run at least three times at that pH using fresh measured solutions. Identify any anomalous result using the pattern and method notes, then calculate a mean from the valid repeats.
8. Repeat the whole method for every pH. Use a fresh dropping pipette, or rinse and dry it thoroughly, between runs so reaction mixture from one pH cannot contaminate the next. Keep all controlled variables and the order of adding solutions the same.

Record raw times before calculating anything:

Plot pH on the horizontal axis and mean relative rate on the vertical axis. The pH with the greatest measured rate is the best estimate of the optimum from those tested values. It is evidence for a pattern, not proof that the exact optimum equals that whole-number pH; smaller pH intervals around the peak would refine the estimate.

Safety and quality of evidence

• Iodine solution can irritate eyes and stain skin or clothing. Wear eye protection, use small drops and rinse splashes promptly.
• Hot water and hot glass can cause burns. Keep the bath stable, avoid splashes and use suitable handling equipment if glass is hot. For the Bunsen-heated beaker bath, use a heatproof mat, secure hair and loose clothing, and switch off the flame when heating is complete.
• Enzyme powder can be harmful if inhaled. Use technician-prepared dilute amylase solution rather than handling powder. Follow supplier/CLEAPSS information for the chosen buffers because their hazards vary.

Repeats reveal random variation and make the mean more reliable; they do not correct a consistently biased method. The 10-second sampling interval limits time resolution because the true endpoint lies within an interval. Sampling every 5 seconds near the endpoint would narrow that interval. Colour judgement is also subjective, so using the same observer, equal drop sizes and fixed reference wells makes comparisons more consistent. Monitoring the water bath matters because a temperature change would affect enzyme activity and confound the effect of pH.

Calculating enzyme activity

A rate compares the change measured with the time taken. An average rate can use substrate consumed or product formed.

If $6.0\,\mathrm{mg}$ of product forms in $30\,\mathrm{s}$, the average rate is $6.0/30=0.20\,\mathrm{mg}\,\mathrm{s}^{-1}$. At that average rate, $2.0\,\mathrm{mg}$ forms in $10\,\mathrm{s}$, which checks the size of the answer.

When every trial reaches the same fixed endpoint from the same initial amount, a shorter time means a faster reaction. For the amylase method, calculate a relative rate:

The unit is $\mathrm{s}^{-1}$, read as “per second”. This is not a mass-per-time rate: no mass of starch was directly measured. Keep the starting amount and endpoint the same for comparisons.

Worked example

At one pH, endpoint times are $48\,\mathrm{s}$, $50\,\mathrm{s}$ and $52\,\mathrm{s}$.

A run taking $100\,\mathrm{s}$ has relative rate $0.010\,\mathrm{s}^{-1}$: twice the time means half the relative rate. Do not call a longer endpoint time a greater activity.