Required Practical 1: Investigation into the effect of a named variable on the

rate of an enzyme-controlled reaction 

Website: Notes - RP 01 Rate of an Enzyme Controlled Reaction - AQA Biology A-level
(physicsandmathstutor.com)

Date accessed: 13/11/22

The rate of reaction of an enzyme-controlled reaction is influenced by different factors: the

temperature, pH, concentration of the substrate, and the concentration of the enzyme. The effect of
each of these can be determined by changing a single variable and measuring its effect on the rate of
reaction. It is important to keep all other variables constant so that they do not influence the results.

Equipment list

● Powdered milk suspension- the Casein in milk break down, the smaller molecules become soluble

● Trypsin solution (0.5%)-  Trypsin has this effect on the rate of the reaction because increasing the
concentration increases the number of particles that can react each second as more enzyme
molecules are available to collide with the protein molecules.

● Distilled water- Distilled water because it does not contain impurities like dissolved ions.

● Hydrochloric acid (0.1M)- This control indicates the colour of a completely hydrolysed sample.

● 5cm3 syringes- To add the milk suspension to each tube

● Flat bottomed tubes- To make it easier to place in the water bath

● Water bath- Used to incubate samples at a constant temperature over a long period of time

● Timer- Measure times accurately

Method 1
In this method the named variable is temperature:

1. Make two control samples:

● Take two flat bottomed tubes.

● Add 5cm3 of milk suspension to each tube.

●Add 5cm3 of distilled water to one tube- this control will indicate the absence of enzyme activity.
● Add 5cm3 of hydrochloric acid to the other- this control indicates the colour of a completely
hydrolysed sample.

2. Take three test tubes and measure 5cm3 milk into each. Place in water bath at 10°C for 5 minutes
to equilibrate.

3. Add 5cm3 trypsin to each test tube simultaneously and start the timer immediately.

4. Record how long it takes for the milk samples to completely hydrolyse and become colourless.

5. Repeat steps 2-3 at temperatures of 20°C, 30°C, 40°C and 50°C.

6. Find the mean time for the milk to be hydrolysed at each temperature and use this to work out
the rate of reaction.

Risk assessment

Hazard Risk Safety precaution In emergency Risk level

Broken glass Cuts from sharp Take care when Elevate cuts; Low
object handling glass apply pressure;
objects; keep do not remove
away from edge glass from
of desk wound; seek
medical
assistance
Hydrochloric acid May cause Wear eye Wash off skin Low
harm/irritation to protection; avoid immediately;
eyes or in cuts contact with skin, flood eye/cuts
tie up long hair with cold water
Hot liquids Scalding Handle with care; Run burn under Low
use tongs to cold water; seek
remove boiling medical
tubes from water assistance
bath; wear eye
protection, keep
away from the
edge of the desk
Enzymes Allergies Avoid contact Seek assistance Low
with skin/eyes;
wear eye
protection

Graph

● Plot a graph of rate of reaction against temperature.

Conclusion

● Milk contains a protein called casein which, when broken down, causes the milk to turn colourless.
Trypsin is a protease enzyme which hydrolyses the casein protein.

As the temperature increases from 10°C, kinetic energy increases so more enzyme-substrate
complexes form. This means that the rate of reaction increases up to the optimum temperature.

● At temperatures beyond the optimum, bonds in the enzyme tertiary structure break, which
changes the shape of the active site. This means that the substrate and enzyme are no longer
complementary..
Required Practical 1: Investigation into the effect of a named variable on the
rate of an enzyme-controlled reaction 
Method 2
The effect of temperature on the rate of the reaction catalysed by trypsin  
The aim of the experiment is to Investigate into the effect of a named variable on the
rate of an enzyme-controlled reaction. temperature, pH, concentration of the
substrate, and the concentration of the enzyme (which in this specific experiment is
the effect of temperature). The effect of each of these can be determined by
changing a single variable and measuring its effect on the rate of reaction.
 
1. Using a marker pen write an ‘X’ on the glass halfway down one side of each of three test

tubes.  
2. Add 10 cm3 of the solution of milk powder to each of these three test tubes.  
3. Add 2 cm3 of trypsin solution to 2 cm3 of pH 7 buffer in another set of three test tubes.  
4. Stand the three test tubes containing the solution of milk powder and the three test

tubes containing trypsin and buffer in a water bath at 20 °C.  

5. Leave all six tubes in the water bath for 10 minutes.  
6. Add the trypsin and buffer solution from one test tube to the solution of milk powder in

another test tube and mix thoroughly.  

7. Put the test tube back into the water bath.  
8. Repeat steps 6 and 7 using the other test tubes you set up.  
9. Time how long it takes for the milk to go clear.  Do this by measuring the time taken to

first see the ‘X’ through the solution.  

10. Record the time for each of the three experiments. 

11.Using the same method, find out how long it takes the trypsin to digest the protein in the
solution of milk powder at 30  
 12.30°C, 40 °C, 50 °C, 60 °C 

Equipment list:
 Test tubes- To place the solution of milk powder and trypsin solution
 Milk powder- the Casein in milk break down, the smaller molecules become
soluble
 Trypsin solution- trypsin has this effect on the rate of the reaction
because increasing the concentration increases the number of particles that can
react each second as more enzyme molecules are available to collide with the
protein molecules.
 PH7 buffer- The shortest time for the starch to disappear is at pH 7
 Water bath- Used to incubate samples at a constant temperature over a
long period of time
 Timer- Measure times accurately
 Thermometer- An accurate piece of apparatus to measure temperature
 Spatula- To mix the trypsin and buffer solution

Risk assessment
Website: CLEAPSS- Enzymes page 33 Date accessed : 13/11/22