Investigating how PH Affects the Activity of Potato Catalase
The aim of this experiment is to investigate how PH affects the
activity of potato catalase. This enzyme occurs in many plant and
animal tissues. Its role is to break down toxic hydrogen peroxide (a
metabolic waste product) into water and oxygen
Catalase catalyses the following reaction:
[IMAGE]2H2O2(l) 2H2O(l) + O2(g)
Oxygen is the only gas given off in the experiment so this will be
measured when the hydrogen peroxide is broken down at different PHs.
Therefore the dependant variable
in the experiment is the amount of
oxygen released and the independent variable is the PH environment of
the catalytical reaction.
The rates of most enzyme catalysed reactions depend on the PH of the
medium in which they occur. Each enzyme is most active at a particular
PH. Its activity
should decrease as the solution is made more acidic
or more basic than its ‘ideal’ PH.
This is due to the ionization of carboxyl, amino and other groups on
either the substrate or the enzyme. In neutral or basic solutions,
carboxyl groups (– COOH) release H+ ions to become negatively charged
carboxylate groups (– COO-). Similarly, amino groups (– NH2 ) accept H+
in neutral or acidic solutions becoming positively charged – NH3+
groups. Thus in a neutral solution, a molecule
with an amino group
will be attracted electrically to another molecule that has a carboxyl
group, because both groups are ionized and they have opposite charges.
If the PH changes, however, the ionization of these groups may change.
For example, at a low PH (high H+ concentration) the excess H+ ions
may react with the – COO- to form COOH. If this happens, the group is
no longer charged and cannot interact with other charged groups in the
protein, so the folding of the protein is altered. If this occurs at
the active site of an enzyme, the enzyme may no longer have the
correct shape to bind to its substrate.
The PH environment of the reaction can be altered and maintained using
a ‘buffer solution.’ This can be added with the hydrogen peroxide.
The various PHs of the buffer solutions will be PH1 (strong acid), PH4
(moderate acid), PH10 (moderate alkalis), PH14 (strong alkalis). The
buffer solution for the control of the experiment will be substituted
with 5cm3 water as water has a neutral PH of 7.
The experiment must be set up as shown below.
· Measure using a measuring cylinder 10cm3 H2O2 and 5cm3 of the
appropriate buffer solution to test tube A
· Dice a raw potato using a sharp bladed knife into ruler measured 1cm3
sided cubes so mass, volume, surface area and shape remain constant in
every cube. The cubes must be from the same potato and must not
include and part of the skin as the enzyme concentration may be
· Ensure the trough is not overfilled, as there should be space for
the displaced water. The increase of water in the trough indicates how
much O2 has entered the test tube.
· Ensure that test tube B is clamped firmly into position as any
movement could distrupt the oxygen flow into the tube.
· Add using a tweezer one of the cubes of potato to test tube A
· Insert a bung into test tube A
· Time using a stopwatch the reaction for ten minutes
· After this time has passed, using the marked scale on the trough,
record the increase in water to identify the amount of oxygen obtained
in the reaction.
· Release Test tube B out of the trough vertically and place a glowing
splint under the test tube (as shown below). The splint should relight
with the presence of oxygen.
To make the experiment fair test the following factors must remain
* Temperature: This must not change. An increase gives the molecules
more kinetic energy so collisions between enzyme and substrate are
more frequent and so the rate of reaction increases.
* Surface area (as well as mass, shape and volume): These must
remain constant. An increase in surface area would enable more
enzymes to react with the substrate (hydrogen peroxide).
* The experiment will be repeated 10 times for each PH and the mean
average will be recorded.
* Ensure goggles are worn at all times as toxic hydrogen peroxide is
* Gloves should be worn when handling H2O2
* Bags and stools should be tucked under desks
* Work surface should be clear in case of any spillages of the
· Lab coats must be worn
* Long hair should be tied back
There are no ethics involved in this experiment as animals or rare
substances are being used in the experiment.
Table of results
PH of buffer.
Amount of O2 released in reaction (millilitres)
*Control of experiment