Part 1: What this practical is testing

This required practical is about measuring how one chosen factor changes the rate of an enzyme-controlled reaction. In AQA's standard example, the named variable is temperature and the reaction is the breakdown of casein in milk by the protease trypsin.

Casein makes the milk suspension cloudy. As trypsin digests the casein, the mixture becomes clearer. That gives us a simple way to judge progress: when an X marked on the tube becomes visible, enough casein has been broken down for the suspension to look clear.

The important biological idea is that rate depends on successful collisions between enzyme and substrate. Below the optimum temperature, warming the mixture increases kinetic energy, so enzyme-substrate complexes form more often. Above the optimum, the enzyme begins to denature, so fewer active sites keep the correct shape.

For this practical, the named variable could be temperature, pH, enzyme concentration, or substrate concentration. The standard trypsin-casein method matters because it teaches the same practical logic you would use for any of those variables.

Part 2: Core method

The AQA handbook method uses these solutions:

• 10 cm^3 of 3% milk solution in each substrate tube
• 2 cm^3 of 0.5% trypsin solution plus 2 cm^3 of pH 7 buffer in each enzyme tube
• temperatures of 20, 30, 40, 50 and 60 degrees C

A clear step-by-step method is:

1. Draw an X halfway down the side of each of three milk tubes.
2. Add 10 cm^3 of milk solution to each of those tubes.
3. In another three tubes, add 2 cm^3 of trypsin and 2 cm^3 of pH 7 buffer.
4. Place all six tubes in a water bath at the chosen temperature and leave them for 10 minutes so both solutions equilibrate.
5. Pour one trypsin-buffer mixture into one milk tube, bung the tube, invert it about five times, return it to the water bath, and start timing immediately.
6. Stop the timer when the X first becomes visible.
7. Repeat for the other two pairs of tubes at the same temperature.
8. Repeat the full set at the other temperatures.
9. Record all raw times, calculate a mean time for each temperature, then convert mean time to rate using rate = 1 / time.

The details matter. If only the milk is placed in the water bath, the reaction temperature is not actually the one you planned. If the tube is shaken too hard, bubbles make the mixture look cloudier than it really is. If the mixture is not inverted in the same way each time, the start of the reaction is not standardised.

Some classes collect data by sharing temperatures between groups and pooling results. That is acceptable as long as the method is consistent.

Part 3: Variables, controls and safety

In the standard version of the practical:

• Independent variable: temperature of the reaction mixture
• Dependent variable: time taken for the milk to clear, or the rate calculated from that time
• Controlled variables: pH, enzyme concentration, substrate concentration, total volumes, equilibration time, and mixing method

The controls need to be stated precisely in exam answers.

• Keep pH constant by adding the same pH 7 buffer each time.
• Keep enzyme concentration constant by using the same trypsin solution and the same volume in every tube.
• Keep substrate concentration constant by using the same milk concentration and volume in every tube.
• Keep equilibration time constant by leaving every set of tubes in the water bath for the same length of time.
• Keep the mixing method constant by using a bung and inverting the tube about five times each run.

A useful negative control is a tube containing milk plus water instead of trypsin. It should remain cloudy. That shows any clearing in the experimental tubes is due to enzyme activity rather than the milk simply settling or changing on its own. PMT notes also describe a comparison tube with hydrochloric acid to show the appearance of a fully hydrolysed sample.

Safety points from the local notes are straightforward but important:

• Trypsin is a protease, so it can irritate skin and eyes and may trigger allergic reactions in sensitive people.
• Eye protection should be worn.
• Splashes should be washed off skin immediately.
• Hot water baths can cause scalds, especially above 50 degrees C.
• Glassware should be handled carefully to avoid cuts.

Part 4: What the results should show

At low temperatures, the reaction is slow because enzyme and substrate molecules move relatively slowly and collide less often. As temperature rises, the rate increases because more collisions are successful. With trypsin, the fastest rate is usually around 40 degrees C in school practical data.

The AQA handbook sample results show the pattern clearly:

• 25 degrees C: mean time 189 s
• 40 degrees C: mean time 79 s
• 50 degrees C: mean time 108 s
• 60 degrees C: mean time 271 s

That is the pattern you should expect to explain in an exam. The reaction speeds up to an optimum, then slows because the enzyme denatures. Denaturation changes the shape of the active site, so the substrate is no longer complementary.

Feynman check

Why is 60 degrees C slower than 40 degrees C even though particles have more energy at 60 degrees C? Because two things are happening at once. Higher temperature increases collision frequency, but once the enzyme starts losing its active site shape, fewer enzyme molecules can bind the substrate at all. Above the optimum, loss of working active sites matters more than the extra kinetic energy.

A good exam habit is to say the enzyme is denatured, not killed, and to link denaturation to changes in tertiary structure and active site shape.

Part 5: Processing the data

Your raw data are times in seconds. From those raw times, you should:

1. Record every repeat in a table.
2. Look for any anomalous result.
3. Calculate a mean time.
4. Convert mean time to rate.
5. Plot rate against the named variable.

The rate calculation is simple:

rate = 1 / mean time

If the mean time at 40 degrees C is 79 s, then:

rate = 1 / 79 = 0.0127 s^-1

When deciding whether a result is anomalous, do not remove it just because it is the highest or lowest value. Exclude it only if it is clearly inconsistent with the other repeats and you have a sensible practical reason, such as poor mixing, bubbles, a temperature drift, or a timing error. Keep the anomalous value in the raw table and make it clear that it was excluded from the mean.

For the graph:

• Plot the independent variable on the x-axis.
• Plot rate on the y-axis.
• Use a line graph because the independent variable is continuous.
• Draw a smooth curve rather than joining dots with a ruler.

If a colorimeter is used instead of the X method, the reading is absorbance after a fixed time. The AQA handbook notes that the trypsin solution can be used as the blank. Lower absorbance means more casein has been digested, so the mixture is less cloudy.

Part 6: Evaluation and transfer to other variables

The biggest weakness in the basic method is that the endpoint is judged by eye. Different students may decide the X becomes visible at slightly different times. That reduces reliability. A colorimeter improves this by giving an objective absorbance reading.

Other common sources of error from the local notes are:

• Water bath temperature changing during the run
• Trypsin deteriorating if it was prepared too early
• Frothing or bubbles if the tube is shaken too vigorously
• Small differences in measured volumes between tubes

The most useful improvements are therefore to:

• use a thermostatically controlled water bath
• prepare fresh enzyme close to the lesson
• use a colorimeter for quantitative readings
• increase repeats and calculate a mean

The same design can be transferred to other named variables.

• For pH, keep temperature constant but use buffers of different pH values.
• For enzyme concentration, change the concentration of trypsin but keep substrate concentration and total volume constant.
• For substrate concentration, change the concentration of milk suspension but keep enzyme concentration constant.

In every case, the principle stays the same: change one variable, control the rest carefully, measure the effect on rate, and explain the pattern using enzyme-substrate collisions and active site shape.