To investigate how enzyme concentration can affect the rate of reaction, in this case the breakdown of milk protein by trypsin, a protease enzyme.

Procedure

In this experiment, the rate of reaction is determined by the time taken for a given quantity of substrate to be used up:

1. Prepare trypsin solutions of different concentrations by mixing trypsin and distilled water together. 2 syringes are used to measure and transfer different volumes of trypsin and distilled water seperately. Label the test tubes:

Test tube A – 2cm3 distilled water

Test tube B – 0.5cm3 trypsin + 1.5 cm3 distilled water

Test tube C – 1cm3 trypsin + 1cm3 distilled water

Test tube D – 1.5cm3 trypsin + 0.5cm3 distilled water

Test tube E – 2cm3 trypsin

2. Tap the bases of the test tubes to mix the enzyme-water solutions.
3. Use another syringe to measure 2cm3 milk and transfer into each of the 5 test tubes. Start each stopwatch once milk is added into each test tube.
4. Tap the bases of the test tubes to mix the solutions.
5. Measure the time needed for the milk to turn clear in each test tube.

Variables

Test tube A is a control.

Explanation: Test tube A contains distilled water only. It ensures that the clearing of milk in other tubes is due to enzyme activity only and not any other factor.

Risk and safety precautions

Take care handling glassware.

Results

Two graphs are plotted.

Graph A: time against concentration

Graph B: reaction rate(=1/T) against concentration

Analysis

Graph A – The graph, which is a downward curve, becomes less steep with increasing concentration:

1. The time taken for the reaction to complete decreases rapidly as concentration of trypsin increases from 0% to 50%.
2. Time taken continues to decrease, but at a slower rate, as concentration of trypsin increases from 50% to 100%.

Graph B – Gradient of the graph is roughly constant.

The points all lie roughly near a straight trendline.

Conclusion

1. The higher the concentration of trypsin, the quicker the milk turns clear, i.e. the higher the rate of breakdown of milk protein.

Reason: the chance of collision between trypsin and milk protein increases. Milk proteins enter the active sites of trypsin to form enzyme-substrate complex at a higher rate. The quicker clearing of a given quantity of milk indicates that milk proteins are used up in the enzyme reaction at a higher rate.

2. Increase in reaction rate is proportional to increase in trypsin concentration.

Reason: increase in the number of trypsin molecules results in a proportional increase in the number of enzyme-substrate complexes and therefore a proportional increase in the rate of reaction.

Limitations and error

1. This experiment does not measure the initial rates of reaction because the quantity of product formed in a fixed period of time from the breakdown of milk protein is not easily observable. Instead, average rates of reaction are measured by determining the time needed for all milk protein to react, i.e. for the reaction to complete. A graph of average rate of reaction against trypsin concentration does not show an accurate straight line.

2. Tapping and shaking the test tubes too vigorously added kinetic energy to the trypsin and milk proteins, making temperature, the controlled variable, not constant.

Improvement

Shake the test tubes slowly.