Title: Investigating the Effect of Substrate Concentration on Enzyme Activity

Aim: To investigate the effect of hydrogen peroxide (H2O2) substrate concentration on the
activity of enzyme catalase

Apparatus and Materials:

 Cork borer 
 Ruler  Measuring cylinder
 Forceps  Syringe (5cm3)
 Boiling tube  Stop-clock
 Rubber bung and delivery tube  Potato tuber
 Test tube  H2O2 solutions ( 0.5, 1.0,1.5 and 2,0
 Beakers mol/dm3)

Diagram:

Diagram showing apparatus used in experiment to observe the effect of different hydrogen
peroxide concentrations, on the enzyme catalase
Procedure:
 A cylinder of potato tuber tissue 0.5cm in diameter and 6cm long was cut out with a cork
borer. The cylinders were sliced into 2mm thick discs and were placed in a water filled
petri dish. Sixty (60) discs were required.
 10cm3 of 0.5 mol.dm3 hydrogen peroxide solution was placed into a boiling tube, using a
syringe. The bung was replaced on the boiling tube. A second tube with tap water was set
up where the delivery tube was 2cm below the surface of the water.
 Ten (10) discs were counted out and all were added to the boiling tube at the same time.
The bung was replaced immediately and the boiling tube was shaken gently to separate
the slices. The numbers of bubbles were counted for one minute after ten seconds had
passed.
 Values were recorded in an appropriate table.
 The contents of the boiling tubes were discarded and the tube was rinsed with cold water.
The procedure was repeated using ten (10) fresh discs and two further readings were
obtained and recorded.
 The procedure was repeated using three other hydrogen peroxide concentrations and
three readings were taken for each solution, and their readings were recorded.
 The mean or averages of the three readings were calculated for each concentration of
hydrogen peroxide.
 A graph was plotted of the average number of bubbles evolved at different
concentrations.

Results:
Bubbles per Concentration of hydrogen peroxide solution H2O2 (mol/dm3)
minute

0.5 1.0 1.5 2.0

Count 1 3 3 7 11
Count 2 2 6 7 9
Count 3 1 1 5 10
Average/Mean 2 3 6 10
Table showing average number of bubbles evolved from reacting different
concentrations of hydrogen peroxide with the enzyme, catalase
Discussion:
Enzymes are globular proteins, which are biological catalysts, which simply mean that they are
made by cells which speed up chemical reactions but undergo no change when the reaction
finishes. Due to this special property of enzymes, only a small amount is used for reactions in
order to be effective.
In the lock and key theory, enzymes work by catalyzing (speeding up) reactions by
combining with the substrate of a reaction. This is possible due to the substrate fitting
into the active sites of the enzyme which are known as clefts or pockets, that hold the
substrate in the active site using various hydrogen and ionic bonds, to form the enzyme
substrate complex. The substrate is the form in which the enzyme is acting upon. In the
modified theory, it is also said that the active site of the enzyme changes shape to
correctly be able to compensate for the shape of the substrate, forming the enzyme
substrate complex.
In every reaction, there are reactants at the start and products at the end. By a number of
intermediate steps, an intermediate molecule is formed usually in metabolic reactions.
The intermediate molecule with the most energy is called the transition state. This
transition state is more stable than a transition state in an uncatalysed reaction (without an
enzyme). The energy needed to convert reactants into products is called activation
energy. An enzyme reduces the amount of activation energy needed for this conversion to
occur. Intermediate molecules contain more energy and are constantly formed until a
final product is formed.
Enzyme activity and the rate of enzyme activity are affected by pH, enzyme
concentration, temperature, incubation time, substrate concentration and the presence of
co-factors or inhibitors.

In this experiment, the substrate, hydrogen peroxide was used in different concentrations to
investigate the effect on the enzyme, catalase.

In the graph plotted, a small gradient was observed as the number of bubbles steadily
increased from two bubbles at 0.5mol/dm3 hydrogen peroxide, to 3 bubbles at 1.0mol/dm3
hydrogen peroxide. A larger slope or gradient was observed on the graph as the number
of bubbles increased to six, at 1.5mol/dm3 hydrogen peroxide. Finally, the largest incline
was observed as a steep gradient was formed as the number of bubbles increased from six
to ten bubbles at 2.0mol/dm3 of hydrogen peroxide.
As the substrate concentration, hydrogen peroxide, increased, the rate at which the
bubbles evolved from each test tube also increased hence a greater number of bubbles
were observed as the concentration of hydrogen peroxide increased. The rate of the
enzyme in the catalysed reaction increased to a point of ten bubbles, called Vmax.
 Each catalase enzyme present within the potato cell contained an active site, the more
concentrated the substrate, hydrogen peroxide, the more substrate molecules present.
These molecules fit into the active sites and fill them and thus more products were
formed. In the test tube, the substrate molecules (hydrogen peroxide molecules) fit into
the active sites of catalase and sped up the breakdown of hydrogen peroxide into water
and oxygen gas. The delivery tube was used to carry the across the oxygen gas into the
water into the second tube where the bubbles evolved were oxygen gas bubbles.
In the experiment carried out, it was observed that the rate of reaction increased as the
substrate concentration (hydrogen peroxide) increased due to the increased rate of the
number of substrate molecules that filled the active sites of the enzymes in the test tube.

In the procedure, 10 discs were used to ensure enough active sites in the enzymes were available
for the substrate molecules to collide with. It was also used as a constant variable used
throughout three separate reactions of the same experiment to obtain the average number of
bubbles evolved between the reaction of different concentrations of the hydrogen peroxide and
the enzyme in the potato discs.
The potato cylinders were cut into discs and allowed a faster reaction of substrate
molecules to react with each disc as the surface area of the potato was increased when it
was cut. This means more substrate molecules over time, were able to attach to the active
sites of the enzyme.
The potato discs and hydrogen peroxide solution were renewed each time because the
active sites in the enzyme of the potato discs were filled and the substrate solution,
hydrogen peroxide already decomposed into water and oxygen gas, hence it would be
unreactive if used again. New substrate solution and potato discs contained empty active
sites that were to be filled again by the substrate.

If twice the number of discs were used in 2mol/dm3 of hydrogen peroxide, more active sites
would be open for the substrate molecules to fill and bubbles would evolve faster and in greater
numbers as the rate at which the hydrogen peroxide decomposed would be increased.

In the time taken for the reaction to occur (one minute), increasing the number of active
sites would increase the amount of products produced, hence more water and more
oxygen gas, seen as bubbles would be produced. Therefore information on how the rate
of enzyme activity is affected by enzyme concentration would be gained about the
reaction.
Precautions:
 Healthy potato was used to ensure the enzyme was present and still functional.
 Test tube was shaken as said in procedure and ensured the contents were mixed
thoroughly to allow the substrate molecules to collide with the active sites of the enzyme.
 When the hydrogen peroxide was measured, in a measuring cylinder, parallax error was
avoided.

Sources of Error:
 Unknown concentration of catalase due to the varying amounts.
 Catalase varies in amount due to uneven cut of potato cylinder, experimental error
due to competence of student.
 Reaction time when counting bubbles- Human Error
 Miscounting bubbles formed in the test tube- Human Error

Limitations:
 Some of the oxygen gas escaped while the potato discs were being placed into the
hydrogen peroxide as the cork was not placed on the test tube.
 Water produced after the reaction could not be measured.

The experiment could have been improved by sealing the test tube with petroleum jelly to
prevent the loss of any oxygen gas. Potato extract could have been used instead of potato discs as
a larger surface area would be available for the substrate molecules to produce products at a
faster and larger rate. Using a gas syringe to measure the amount of oxygen gas produced would
be more accurate than counting the number of bubbles as measuring with the gas syringe
involves fewer errors compared to observing the number of bubbles produced which was prone
to human error.

Conclusion:
As the concentration of hydrogen peroxide increased from 0.5 to 2.0 mol/dm3, the rate of
reaction increased from 2 bubbles to 10 bubbles per minute. There was an increasing rate
of reaction, which became constant with Vmax at 10 bubbles per minute.