Example Activity

The rate of reaction describes how quickly substrate disappears or product forms:

$\text{rate}=\frac{\text{change in amount of substrate or product}}{\text{time}}$

Temperature, pH and substrate concentration can all affect this rate. An investigation changes one of these factors while controlling the others.

A practical investigation: pH and amylase

Amylase catalyses the breakdown of starch into maltose. Iodine solution provides a way to detect the starch:

• iodine remains orange-brown when starch is absent;
• iodine turns blue-black when starch is present.

The reaction mixture can therefore be sampled repeatedly. The first sample that leaves the iodine orange-brown shows that no starch is detected and the chosen endpoint has been reached.

Use amylase solution, starch solution, labelled buffer solutions of different pH, iodine solution, test tubes, measuring apparatus, pipettes, a spotting tile, a stopwatch and a water bath. Wear eye protection because iodine solution is an irritant, and follow the assessed precautions for the heating equipment used.

A suitable method is:

1. Put one drop of iodine solution into each of several wells of a spotting tile.
2. Measure fixed volumes and concentrations of amylase and starch. Use a buffer to set the first pH.
3. Bring the reactants to a fixed temperature in a water bath. This prevents temperature changes from confusing the effect of pH.
4. Combine the reactants, mix consistently and start the stopwatch immediately.
5. At fixed intervals, such as every 10 seconds, transfer one drop of reaction mixture to the next iodine drop. Use a clean pipette or an uncontaminated sampling method.
6. Record the time at which the iodine first remains orange-brown.
7. Repeat with different buffer pH values, keeping the temperature, volumes, concentrations and sampling interval constant.
8. Repeat each pH and calculate a mean, checking any anomalous result before excluding it.

Do not add the iodine directly to the main reaction mixture: it is the test reagent, not a reactant whose concentration should change during the investigation.

Variables and a fair comparison

The independent variable is pH. The dependent variable is the measured reaction time or a rate calculated from it. Important control variables include temperature, amylase concentration and volume, starch concentration and volume, total reaction volume, mixing and the sampling interval.

Because every trial begins with the same amount of starch and ends at the same endpoint, a shorter time means a faster reaction. A useful relative rate is:

$\text{relative rate}=\frac{1}{\text{completion time}}$

If time is measured in seconds, the relative-rate unit is $\text{s}^{-1}$. This is an inverse relationship: doubling the time halves the relative rate.

Interpreting pH results

Plot pH on the horizontal axis and rate on the vertical axis. The highest point estimates the enzyme's optimum pH. If completion time is plotted instead, the optimum is represented by the lowest point.

Data for Illustrative effect of pH on amylase activity

Changing pH can alter the forces maintaining an enzyme's three-dimensional shape. Away from the optimum, fewer active sites bind substrates successfully and the rate falls. At sufficiently extreme pH, the active site changes shape and the enzyme may become denatured. An optimum belongs to a particular enzyme under particular conditions; it is not correct to assume that every enzyme has the same optimum pH.

Investigating temperature instead

The same starch–amylase indicator method can be adapted by changing temperature rather than pH. Place separate tubes of starch and amylase in a water bath at the chosen temperature long enough for both to reach it. Combine them, start timing and test samples with iodine at regular intervals. Repeat at several temperatures while keeping pH, concentrations and volumes constant.

At low temperatures, particles have less kinetic energy, so successful collisions between enzyme and substrate happen less frequently. Warming initially increases the rate. Above the optimum, heat disrupts the enzyme's structure; its active site changes and the rate falls rapidly as the enzyme denatures. Cooling slows an enzyme but does not itself mean that the enzyme has been denatured.

Investigating substrate concentration

Substrate concentration may also be varied while enzyme concentration, pH, temperature and total volume are controlled. At low substrate concentrations, increasing concentration produces more frequent enzyme–substrate encounters, so the initial rate rises. Eventually, nearly all active sites are occupied most of the time. Enzyme concentration then limits the reaction and adding more substrate produces little or no further increase in rate.

When substrate concentration changes, simply comparing the time needed to use up all the substrate can be misleading because different trials begin with different quantities. Instead, compare initial rates using a method that measures the same defined change in each trial.

Improving the evidence

Continuous sampling gives only an estimated endpoint. If samples are taken every 10 seconds, the true endpoint lies somewhere between the final blue-black sample and the first orange-brown sample. Shorter, accurately timed intervals improve time resolution, although they make the procedure harder to perform consistently.

Repeat measurements reveal random variation and allow a mean to be calculated. A thermostatically controlled water bath improves temperature control, buffers maintain pH, and calibrated measuring apparatus makes reactant volumes more consistent. These improvements make it more likely that any pattern is caused by the chosen independent variable rather than another changing condition.