Core Practical 4: Factors Affecting Enzyme-catalysed Reactions

01Follow a changing signal

The supplied Pearson practical uses a protease such as trypsin to break down casein in a milk suspension. As the suspension clears, less light is scattered and the instrument’s apparent absorbance falls; transmission rises. The magnitude of the initial absorbance gradient is an activity proxy.

Use a colorimeter or datalogger, matched cuvettes, measured milk and enzyme solutions, buffers, water baths, thermometer, pipettes and timer. The teacher selects safe reagent strengths and a pilot range. An optical change is not automatically a molar reaction rate; converting it requires an appropriate calibration.

02A repeatable measurement sequence

Prepare measured enzyme and substrate portions separately, allowing them to reach the chosen temperature before mixing. Use a suitable blank containing the assay components except milk, with milk replaced by water. Keep total reaction volume and optical path consistent.

Add enzyme last, mix consistently and begin timing immediately. Record apparent absorbance at short regular intervals from the start, choosing intervals fine enough to resolve the initial change. Maintain reaction temperature during measurement where possible, or measure and report drift. Clean cuvettes between runs and repeat with freshly prepared reactions.

03Investigate temperature and pH separately

For temperature, use a teacher-approved range of water baths and measure actual temperatures. Keep final enzyme concentration, substrate concentration, pH and volumes fixed. Pre-equilibrate solutions separately so reaction does not begin before timing.

For pH, use a suitable range of buffers at the same added volume and check the final reaction pH. Keep temperature, final enzyme and substrate concentrations and total volume constant. Include a no-enzyme control at each pH if pH alone affects milk cloudiness. The buffer is a control tool, not the substrate.

04Investigate enzyme and substrate concentrations separately

For enzyme concentration, prepare known dilutions and add the same volume from each to equal substrate portions. Keep final substrate concentration, temperature, pH and total volume constant. A stock concentration is diluted again on mixing: 2 cm³ of 1% enzyme made up to 4 cm³ total gives 0.5% final enzyme.

For substrate concentration, vary the milk dilution while keeping enzyme concentration, temperature, pH and total volume fixed. Allow for different starting cloudiness. A common validated relationship between optical signal and substrate amount is needed before comparing chemical rates across different milk concentrations; avoid treating the percentage of starting absorbance lost as automatically equivalent to amount digested.

05Calculate initial rates, then compare factors

Plot apparent absorbance against time for each reaction. Draw a tangent at the start and calculate its gradient using well-separated points on that tangent. Because absorbance falls, the gradient is negative; report its magnitude as a positive activity measure when clearly labelled.

For an illustrative initial tangent falling from 0.90 to 0.66 over 40 s, rate magnitude = 0.24/40 = 0.0060 absorbance units s⁻¹. Calculate each repeat’s initial rate, then a mean and spread for each condition. Plot mean initial rate against the independent variable. Do not average an entire curved time-course and call it the initial rate.

06Interpret trends and limitations

Temperature can increase activity through molecular motion until structural instability reduces activity; pH can change active-site interactions and protein structure. More enzyme supplies more active sites when substrate remains sufficient; more substrate increases rate until available enzyme capacity becomes limiting.

Mixing delays, temperature drift, bubbles, scratched cuvettes, settling milk and poorly resolved early readings can distort the initial gradient. Pilot a slower assay or use faster logging if much of the reaction finishes before the first reading. A no-enzyme control distinguishes spontaneous optical changes from enzyme activity.

07Risk controls

Wear eye protection and avoid skin or eye contact with protease and buffers. Enzymes can irritate or sensitise; staff should prepare stock solutions and handle powders using the school’s controls. Do not taste milk or other laboratory reagents. Handle hot baths and tubes carefully, keep instruments dry and follow the local disposal procedure.