The
decomposition of hydrogen peroxide by the enzyme catalase
In this experiment you are measuring the rate at which oxygen is formed
from the enzyme catalase decomposing hydrogen peroxide.
Here, the product
oxygen gas, provides the means of following the rate of the reaction.
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
For this reaction there are three experimental
situations are fully described
(a)
Changing concentration of substrate molecule or
enzyme
(b)
Changing the temperature of the reaction mixture -
looking for the optimum
(c)
Changing the pH of the reaction mixture - looking for
the optimum
(a) The basic
procedures for method 1.
Method 1. A
method of measuring the rate of product formation from an enzyme reaction
(a)
Investigating the effect
of changing the concentration for an enzyme reaction
(decomposition of hydrogen
peroxide to water and oxygen using the enzyme catalase)
(vary either the hydrogen peroxide or
the enzyme catalase).
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
Mashed up potato made into a fine slurry
diluted with acts as a
source of the enzyme catalase. It needs to be well shaken before use so
that each portion measured out has the same amount of catalase in it.
A slurry is a pulverized solid mixed in
a liquid.
You need a series of hydrogen peroxide solutions
of known different concentrations and a fixed concentration of the potato
mix to investigate the effect of changing the hydrogen peroxide
concentration.
You may have to do some 'trial and error'
experiments to find out which amounts give 'reasonable' results.
You can also keep the hydrogen peroxide
concentration constant and do the investigation with a set of
different concentrations of the potato-catalase mixture.
The water bath is set to a constant temperature
e.g. 25oC. The apparatus is setup as illustrated above.
The optimum conditions for human
catalase are pH 7 (so no need for buffer) and 37oC
If no thermostated water bath is available you can
get reasonable results if the laboratory temperature stays reasonably
constant - but record and monitor the room temperature.
You can use a beaker of heated water but its
difficult to keep it at a constant temperature.
The 'stock' solutions of potato-catalase or hydrogen
peroxide should be initially in separate boiling tubes and placed in the
water bath so that everything starts at the right temperature. Or, if no
water bath available, they can be just put together in test tube racks on
the laboratory bench, but you should monitor and record the room
temperature.
Depending on what accuracy you require, you can
measure out a fixed amount of the potato slurry and a varied amount of
the same hydrogen peroxide solution into the boiling tube using a
pipette, or 10 cm3 measuring cylinder or plastic syringe.
You should keep the total volume of the reaction mixture the same.
There shouldn't be a need for a buffer, but
the mixture should have a constant pH of ~7.
If in doubt build a fixed volume of a pH 7
buffer into your method.
You should make up the reaction mixture of
hydrogen peroxide and potato slurry as quickly as possible and shake
well.
Your reaction mixture to vary the hydrogen
peroxide concentration may be as follows
w cm3 of buffer (if used)
x cm3 of potato slurry - kept
constant
y cm3 of hydrogen peroxide solution
- variable
z cm3 of water - variable
w + x + y + z = total constant volume and
volume y + z must also be kept constant.
You can vary y and z to give different
concentrations if different stock solutions of
By varying volumes y and z you can produce
a range of hydrogen peroxide concentrations if a variety of
stock solutions are not available..
The boiling tube and mixture is quickly connected
to the delivery tube rubber bung and placed in the water bath and the stop watch started.
Make sure the boiling tube is fully immersed in water so it and the
contents are at the right temperature.
Start the stopwatch. You can now measure how much oxygen is formed in a
set time e.g. 1 minute, and repeat the experiment several times with the
same volumes of reactants at the same temperature.
This will allow a
more accurate mean value of the rate of reaction to be used in the final analysis.
(Or set of volume readings for one run over a longer time, and plot graph of volume versus time
and measure the initial gradient, but more work for repeats - see graph
on the right)
However you get the results, the rate is
calculated as follows:
From the initial gradient of the graph, the rate of enzyme reaction is expressed as:
rate = volume
of O2 formed ÷ time taken (cm3/s)
You then
draw a graph of the mean values of the rate of reaction (in cm3/s)
at each temperature versus concentration.
You should find that the rate increases with
increase in either hydrogen peroxide or enzyme concentration, if you
have been very accurate you may
get a nice linear graph like the one on the right or else!
You then repeat the whole exercise with different
concentrations of the enzyme using the kind of x + y + z 'recipe'
described above using a fixed concentration of hydrogen peroxide.
Gas syringe system
It is possible to get more accurate results using a
gas syringe system, as long as the flask can be set up in a water bath
(omitted from the diagram below!) or the laboratory temperature stays
constant.
The investigation is conducted in the same way as
already described above.
You can get more accurate data of the volume of oxygen
formed over time and from the graphs work out the initial rate of reaction
from the initial gradient (see right-hand side of above diagram).
Graph line A (steeper gradient) compared to graph line
B may represent (i) an increase in concentration of either substrate or
enzyme, or (ii) an increase in temperature or (iii) a solution pH nearer the
optimum value for that particular enzyme.
See also GCSE chemistry notes:
Effect on rate of changing reactant
concentration in a solution
(b)
Investigating the effect
of changing temperature
for an enzyme reaction
(decomposition of hydrogen
peroxide to water and oxygen using the enzyme catalase)
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
Mashed up potato made into a fine slurry diluted
with water acts as a
source of the enzyme catalase. It needs to be well shaken before use so
that each portion measured out has the same amount of catalase in it.
You need a hydrogen peroxide solution of known and
constant concentration.
You may have to do some 'trial and error'
experiments to find out which amounts give 'reasonable' results.
You should also use the same volume of the
well shaken potato slurry.
The water bath is set to the start temperature
e.g. 20oC. The apparatus setup is illustrated above.
You could start as low as 10oC
perhaps by cooling the water with ice, not sure how well it would
work?
The two 'stock' solutions should be initially in
separate boiling tubes and placed in the water bath so that everything
starts at the right temperature - solutions and boiling tube.
Depending on what accuracy you require, you can
measure out a fixed amount of the potato slurry and a fixed amount of
the same hydrogen peroxide solution into the boiling tube using a
pipette or a 10 cm3 measuring cylinder or plastic syringe.
You should keep the total volume of reaction mixture constant.
(There shouldn't be a need for a buffer, the mixture should have a constant pH of ~7)
(If in doubt use a buffer to match the optimum
pH of the enzyme catalase).
You make up the reaction mixtures as quickly as
possible in a boiling tube and shake well.
The boiling tube and mixture is quickly connected
to the delivery tube rubber bung and the stop watch started. Make
sure the boiling tube is fully immersed in water so it and the contents
are at the right temperature.
Start
the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1
minute, and repeat the experiment several times with the same volumes of
reactants at the same temperature.
Repeats will allow a more accurate average value
of the rate of reaction to be used in the final analysis.
(or set of volume readings for one run, plot graph of volume versus time
and measure the initial gradient, but more work for repeats - see the
graph on the right)
From the initial gradient of the graph, the rate of enzyme reaction is expressed as:
rate = volume
of O2 formed/time taken (cm3/s)
You then repeat the whole exercise at 30oC,
40oC, 50oC etc. adjusting the thermostat
temperature control.
You should find from 20oC to 40oC
an increase in the rate of oxygen production, but an increasingly slower
rate of reaction from 50oC to 70oC (see graph on
bottom
right).
You then draw a graph of the mean values of the
rate of reaction (in cm3/s) at each temperature versus
temperature.
See the end of method 1. (a) for a
gas syringe method
See also GCSE chemistry notes:
Effect on
rate of
changing the temperature of reactants
(c)
Investigating the effect of changing pH
for an enzyme reaction
(decomposition of hydrogen
peroxide to water and oxygen using the enzyme catalase)
Enzyme reaction equation: hydrogen peroxide ===
catalase ===> water + oxygen (gas)
2H2O2(aq)
====> 2H2O(l) + O2(g)
Mashed up potato made into a fine slurry diluted
with water acts as a
source of the enzyme catalase. It needs to be well shaken before use so
that each portion measured out has the same amount of catalase in it.
You need a hydrogen peroxide solution of known and
constant concentration AND stock solution of the potato slurry to
provide the enzyme catalase.
You may have to do some 'trial and error'
experiments to find out which amounts give 'reasonable' results.
You need a range of at least five stock solutions of buffers giving a
variety of pH values e.g. ideally from pH 2 to pH 11.
A buffer solution keeps the pH constant in a
reaction medium - it can neutralise small amounts of acid or alkali
formed.
The water bath is set to a constant temperature
e.g. 25oC-35oC.
The higher temperature is faster - do a trial
run, if too slow raise the temperature, but don't go above 35oC).
The apparatus setup is illustrated above.
If no thermostated water bath is available you can
get reasonable results if the laboratory temperature stays reasonably
constant - measure and monitor.
The 'stock' solutions of catalase, hydrogen
peroxide and the buffer solutions should be initially in separate
boiling tubes and placed in the water bath so that everything starts at
the right temperature. Or, if no water bath available, they can be just
together in test tube racks on the laboratory bench, but you should
monitor and record the room temperature.
Depending on what accuracy you require, you can
measure out a fixed amounts of the potato slurry, hydrogen peroxide
solution and the buffer solutions into the boiling tube using a pipette
or more accurately with a 10 cm3 measuring cylinder.
Whatever your 'recipe', keep the total volume
of the three solutions constant for the final reaction mixture.
The three solutions are mixed in a boiling tube
and well mixed, the total volume should be constant and you use the same
concentrations of the hydrogen peroxide and potato-catalase. The pH of
the buffer should be the only variable.
The boiling tube and mixture is quickly connected
to the delivery tube rubber bung and the stop watch started. Make
sure the boiling tube is fully immersed in water so it and the contents
are at the right temperature.
Start
the stopwatch. You can now measure how much oxygen is formed in a
set time e.g. 1 minute, and repeat the experiment several times with the
same volumes of reactants at the same temperature.
This will allow a
more accurate mean value of the rate of reaction to be used in the final analysis.
(or set of volume readings for one run, plot graph of volume versus time
and measure the initial gradient, but more work doing repeats - see the
graph on the right)
From the initial gradient of the graph, the rate of enzyme reaction is expressed as:
rate = volume of O2 formed/time
taken (cm3/s)
You then repeat the whole exercise with different pH
buffer solutions.
You then draw a graph of the mean values of the
rate of reaction (in cm3/s) versus the pH, and it 'may' look
like the graph on the right.
See the end of method 1. (a) for a
gas syringe method
Summary of learning objectives and key words or phrases
Know about the experimental methods for investigating the enzyme catalysed decomposition of hydrogen
peroxide by catalase and how to investigate the effects of changing temperature,
changing pH and changing the concentration.
Know what
apparatus is needed and the main points of the experimental procedure for
the enzyme decomposition of hydrogen peroxide, including the apparatus and chemicals required.
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