Investigation into the effect of enzyme or substrate concentration
on enzyme activity
Specification reference:
AS Component: 1.4
A level Component: Core concepts 4
Biological reactions are regulated by enzymes
Introduction
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The substrate concentration in an enzyme catalysed reaction affects how much product is
made.
Catalase is an enzyme that increases the breakdown of hydrogen peroxide, a toxin
produced as a bi-product of reactions in cells. The reaction of catalase is shown below.
2H 2 O 2
R
R
R
R
2H 2 O + O 2
R
R
R
Catalase is found in high concentration in raw potatoes.
Apparatus and reagents
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Freshly cut potato cylinders
Pestle and mortar
Specimen tubes/test tubes
Stock solution of hydrogen peroxide 10 vol ( labelled stock solution
Filter paper discs
Forceps
Stopwatch
Syringe
Distilled water
Paper towel
Method
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1. Grind a 2 cm piece of potato cylinder with 5 cm3 of distilled water to make a smooth
P
P
paste containing the enzyme.
2. Place 10cm3 of H 2 O 2 in a specimen tube/ test tube.
P
P
R
R
R
R
3. Using forceps, dip a filter paper disc into the enzyme suspension, tap off the excess.
4. Drop the filter paper disc into the hydrogen peroxide solution and measure the time,
to the nearest second, that it takes from striking the surface to sink to float up to the
surface again.
5. Remove the disc from the tube using forceps and discard.
Risk assessment
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Hazard
Cork borers are sharp
Risk
may cut skin when cutting
cylinders
Hydrogen peroxide is an
irritant
Hydrogen peroxide may
splash onto the skin or into
the eyes.
Control measure
The cylinders of tissue must
be cut on a white tile with the
force directed downwards.
Wear safety glasses
Teacher/ Technicians notes
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No details of independent variable have been given in this method and students should be
encouraged to plan their own investigation. They should be able to identify all the variables
involved as outlined in the guidance notes at the front of the lab book and also an
appropriate range for the independent variable. If the variables chosen are not appropriate,
then it would be acceptable for teacher to make some amendments before the plan is
carried out.
The hydrogen peroxide solution can be labelled as a stock solution or as a concentration in
vol.
If it is impractical for students to make their own enzyme extracts they could be provided with
homogenised potato extract. This can be substituted with other sources of catalase, e.g.
mung beans.
The filter paper disks can be made using a cork borer or hole punch (5-8mm diameter).
Each student should be supplied with 15 discs of the same size.
Students will need access to safety glasses for this experiment.
Sample results
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Concentration
hydrogen peroxide.
(% stock solution)
10
20
40
60
80
100
Time taken for disc to rise to surface (seconds)
1
130
60
40
9
8
9
2
134
58
44
10
10
10
3
140
62
42
11
8
8
Mean
135
60
42
10
9
9
Rate 1/time
0.007
0.017
0.024
0.010
0.11
0.11
Further work

Extension work could include a comparison of catalase activity from a range of
sources such as mung bean or liver.

The technique could be used to study the effect of substrate or enzyme concentration
on enzyme activity.

Students should be encouraged to calculate the rate of reaction (1/time).
Practical techniques
 use appropriate apparatus to record a range of quantitative measurements (to
include mass, time, volume, temperature, length and pH)
 use laboratory glassware apparatus for a variety of experimental techniques to
include serial dilutions