IGCSE Biology Practical Skills 6: Enzyme Rate Investigations

Rates of enzyme-catalysed reactions, including judged colour endpoints, are an explicit Cambridge IGCSE Biology practical context. Papers 5 and 6 can vary temperature, pH, enzyme concentration or substrate concentration and require a valid rate measure, controlled method, graph and evidence-based evaluation.

Define what rate means in the method

Rate is change in a measured quantity per unit time. A continuous method measures product formation or substrate loss repeatedly, such as oxygen volume over time. An endpoint method measures the time required to reach the same defined state.

When every trial reaches the same endpoint from the same starting amounts, comparative rate = 1 / endpoint time. If time is measured in seconds, the unit is s⁻¹.

Reciprocal time compares rates but is not automatically an exact concentration change per second. State what was measured and avoid claiming a stronger unit than the evidence supports.

Amylase and starch endpoint

Place iodine solution in separate wells of a spotting tile. Equilibrate amylase, starch and any buffer at the selected condition. Mix amylase and starch and start the timer immediately.

At fixed intervals, use a clean pipette to transfer one drop of reaction mixture to a fresh iodine well. Early samples turn blue-black while starch remains. The endpoint is the first sample for which iodine stays orange-brown, indicating that starch is no longer detected.

Use separate pipettes for iodine and reaction mixture to prevent contamination. Keep drop size, sampling interval, volumes, concentrations and endpoint rule constant.

The true endpoint lies between the last positive and first negative sample. Shorter sampling intervals reduce this timing uncertainty but require faster, consistent handling.

Investigate temperature

Prepare water baths across a safe, useful range. Place enzyme and substrate tubes separately in the selected bath long enough to reach that temperature before mixing.

Keep pH, enzyme concentration and volume, substrate concentration and volume, total volume and measurement method constant. Monitor actual mixture temperature rather than relying only on a bath label.

Rate commonly rises with temperature initially because particles move faster and successful collisions occur more often. Above an optimum, denaturation changes the active site and rate falls.

Low temperature slows molecular movement; it does not normally denature the enzyme. Do not infer an exact optimum if tested intervals are wide.

Investigate pH

Use buffer solutions to set a range of pH values. Keep buffer volume and total reaction volume constant. Equilibrate all solutions at the same temperature.