The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.

Introduction:

Enzymes are proteins that speed up many

chemical reactions in cells. They act on
substrates. Each enzyme acts only on specific
substrates. Also it might be a reaction between a
specific combination of subtstrates and a
specific enzyme. Enzymes affect the rate of
almost all chemical reactions which take place
in organisms. Like all other proteins, enzyme
function may be affected by a change in any
aspect of its structure. The effect of a change in
enzyme structure is called denaturation. Some
substrates fit in the active site of the enzymes,
but they are not taken apart by enzymes.They dont let enzyme, which they occupied, take part in
reactions anymore. They are called inhibitors.
The activity of enzymes is affected by pH, by temperature, presence of inhibitors, enzyme
concentration and substrate concentration. There are still large areas of medical research in which
the diagnostic potential of enzyme reactions has not been explored at all.
Catalase is an enzyme which is produced by every cell to break down hydrogen peroxide.
Catalase is located in a cell organelle called the peroxisome. Hydrogen peroxide is a waste product
of cellular activity that is poisonous to cells. However, it was used for a long time as a wound
treatment (Some Eastern European countries still use it). Catalase speeds up the decomposition of
hydrogen peroxide
into harmless water
and oxygen gas.
The purpose of
this lab was to
determine the effect
of
pH
and
temperature on the
rate of catalase enzyme activity. Since the catalase speeds up the decomposition of hydrogen, the
rate of catalase activity was measured indirectly by measuring the change in % oxygen produced
over a fixed time period.
There were
two experiments
perfromed. First
Independent 1 experiment:
pH
2 experiment: one determined
variable
temperature
the dependence
of
rate
of
Controlled
Amount of time
catalase activity
variable
on the level of
pH. In the first
experiment our hypothesis was that if the level of pH decreases, then the rate of hydrogen peroxide
degradation decreases. The second experiment investigated the effect of temperature on catalase
activity. In the second experiment our hypothesis was that if the temperature decreases, then the
speed of hydrogen peroxide decomposition decreases.
Dependent
variable

Catalase enzyme activity (measured as % of oxygen produced)

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.
Dependent
variable

Catalase enzyme activity (measured as % of oxygen produced)

Independent
variable

1 experiment: pH

Controlled
variable

Amount of time

2 experiment: temperature

Materials and Methods:

Since hydrogen peroxide is potentially damaging to skin and eyes, goggles were worn for the
duration of the experiment.
A Vernier Oxygen Gas Sensor probe was connected to a Vernier Logger Pro data collector, the
apparatus was checked to confirm that it was working correctly.
200 ml of catalase solution was prepared by mixing 25 g of COOP yeast into lukewarm distilled
water.
Experiment 1: Effect of pH on the rate of decomposition of hydrogen peroxide:
The first experiment investigated the effect of pH change on the rate of decomposition of hydrogen
peroxide.
(Using a 2 ml measuring pipette), 5 ml of 3% hydrogen peroxide (H 2O2) were measured into 3 25
ml test tubes (labelled pH4, pH7 and pH10).
5 ml of specific pH buffer (pH 4, pH 7 and pH 10) were added to each test tube.
The pH of the buffer/ H2O2 mixture was measured using universal pH indicator.
2 ml of catalase was added to the test tube labelled pH 4, the mixture was transferred into a closed
chamber and a stopwatch was started. 30 s after transfer, measurement of % oxygen was started
using the Vernier apparatus. Data collection was continued for 180 s. This procedure was
repeated for the test tubes containing hydrogen peroxide/pH 7 and pH 10 buffer. Data was saved
and transferred to EXCEL for the purpose of data analysis.
Experiment 2: Effect of temperature on the rate of decomposition of hydrogen peroxide:
The second experiment investigated the effect of temperature change on the rate of decomposition
of hydrogen peroxide.
An ice bath was prepared and maintained at 0 oC. 3% hydrogen peroxide, catalase solution and
distilled water were maintained in the ice bath. Temperature was confirmed using a thermometer.
2

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.
Similarly, a hot water bath was prepared and maintained at 60 oC. 3% hydrogen peroxide, catalase
solution and distilled water were maintained in the hot water bath. Temperature was confirmed
using a thermometer.
(Using a 2 ml measuring pipette), 5 ml of 3% hydrogen peroxide (H 2O2) were measured into 3 X 25
ml test tubes (labelled 0 oC, 22 oC and 60 oC).
5 ml of distilled water were added to each test tube.
2 ml of catalase was added to the test tube labelled 0 oC, the mixture was transferred into a closed
chamber and a stopwatch was started. 30 s after transfer, measurement of % oxygen was started
using the Vernier apparatus. Data collection was continued for 180 s. This procedure was
repeated for the test tubes containing hydrogen peroxide/22 oC and 60 oC. Data was saved and
transferred to EXCEL for the purpose of data analysis.

Results:
Experiment 1:
Table 1:
The effect of pH change on the rate of oxygen produced by breakdown of hydrogen peroxide
Reaction No
No
Rate
peroxide Catalase pH4
pH 7
pH 10
0 - 60 s
0.000
0.001
0.006
0.007
0.019
61 - 120
s
0.000
0.000
0.013
0.011
0.035
121
180 s
0.000
0.001
0.019
0.014
0.029
0 - 180 s 0.000
0.001
0.012
0.011
0.027
Graph 1:
The effect of pH change on the rate of oxygen produced by breakdown of hydrogen peroxide

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.

Graph 2:
The effect of pH change on the rate of oxygen produced by breakdown of hydrogen peroxide

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.
Experiment 2:
Table 2:
The effect of temperature change on the rate of oxygen produced by breakdown of hydrogen
peroxide
Reaction
Rate
0 - 60 s
61 - 120 s
121 - 180 s
0 - 180 s

0C
0.002
0.004
0.008
0.005

22 C
0.021
0.034
0.032
0.029

60 C
0.008
0.005
0.005
0.006

Graph 3:
The effect of temperature change on the rate of oxygen produced by breakdown of hydrogen
peroxide

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.
Graph 4:
The effect of temperature change on the rate of oxygen produced by breakdown of hydrogen
peroxide breakdown

Discussion:
In Experiment 1 it was observed that any change in the level of pH (both decrease and increase)
affected the speed of breakdown of hydrogen peroxide. In Experiment 1 was observed that both
decrease and increase of the level of pH increased the speed of hydrogen peroxide breakdown.
However, the rate of oxygen production was higher at the pH level 10 than at the pH level 4.
Without any catalase or hydrogen peroxide,no oxygen was produced, since there was no t hydrogen
peroxide breakdown. Our hypothesis was refuted. We assumed that the higher the pH, the faster the
rate of hydrogen peroxide breakdown. However, the patterns which were assumed were not
obeserved in the experiment. After the experiment was made a conclusion that any change in pH
speeds up the produce of oxygen by breakdwon of hydrogen peroxide, but the increase in pH speeds
up the breakdown process higher, rather than decrease.
In Experiment 2 it was observed that any change in temperature negatively affects the speed of
hydrogen peroside breakdown Not exactly a change in temperature affects the rate of hydrogen
peroxide breakdown. The rate of decomposition of hydrogen peroxide at room temperature was
much greater than at 60 Co and 0 Co. It was assumed before the experiment that the lower the
temperature, the lower speed of hydrogen peroxide decomposition. Our hypothesis was partly
upheld by experimental data.. However, it was observed that any great change in temperature
negatively affects enzyme catalase activity, since at these temperatures enzyme denatures.

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.
Error sources:
In both experiments, more precise and accurate apparatus should be used to measure the volume of
liquids (pH solutions, yeast, hydrogen peroxide), to control the ratio of liquids and the purity of the.
The measuring burette would perfectly fit.
The oxygen measuring apparatus also should be improved,. The devices were not calibrated before
the experiment began, andtheir accuracy was not confirmed. Also it was unknown whether these
devices were calibrated. These factors could affect the data that was collected.
In Experiment 2 to control the temperature of catalase and hydrogen peroxide apart from water and
ice bath, the temperature of the environment (room where exepriment was performed) should also be
controlled, as the catalase and hydrogen peroxide, in order to improve the relaibility of the results
obtained.
Also in each, there was a 30 second delay before oxygen measurement began. . As human error
factor s are present in every experiment, there might be a mistake of 1-2 seconds, which may have
affected the results of the experiment.

Conclusion:
In experiment, 1 was observed that a change in pH alters the rate of

decompositionof

decomposition of hydrogen peroxide. The optimal pH for cyeastyeast catalase activity appears to
be pH 10. It ust be recognized, howebverhowever, that at extreme pH levels (pH 1-2 and pH 1314 )14), the enzyme denatures and the speed of hydrogen peroxide breakdown decreases. In
experiment 2, extremes of temperature reduced the rate of hydrogen peroxide breakdown. It
happened, because at these temperatures hydrogen peroxide denatures.

In fact, at low

temperatures the enzyme should not be denatured, but the reaction rate would be slow since the
rate of enzyme-substrate collisions would be slow.
This is a different font to the rest of the document..

References:
27 July 2004, SEPS. Catalase FAQ.
Repose, D. 2008. Introductory Biology at UNCG. Writing a lab report.
8

The effect of level of pH and temperature on

catalase enzyme activity over oxygen produce.
2009, MHS Science. Sample Lab Report: Factors which Affect the Acivity of the Enzyme Catalase