Effect of pH on

Catalase
By: Morgan Haines, Grace Barton, Brett Cowan, Rachel Urban

Abstract:

The purpose of the report is to state the effect pH has on the enzyme, Catalase. The

enzyme Catalases purpose is to breaks down hydrogen peroxide that can harm the body. To test
the effect pH has on the enzyme Catalase is by measuring the pressure that is produced by the

chemical reaction when the enzyme is placed in a Hydrogen peroxide solution. The way of

comparing results is taking the slope of the pressure line through a 200 second test. The pH of

the hydrogen peroxide solution is changed each trial using pH buffers. As the pH went up during

the experiment the higher the slope was with the exception of one result that could be excluded

or redone for the best results. Using the data that was gathered during the experiment it was

shown the higher the pH was then more efficient the enzyme catalase was at breaking down the

hydrogen peroxide solution.

Background:

The purpose of the experiment that we conducted was to find an answer to this question.

The question is this: How does changing the pH of the hydrogen peroxide solution affect the

efficiency of the catalase enzyme. Before a reliable experiment could be formulated/ conducted,

much research was done to obtain optimal information to help create the hypothesis and

experimental design.

First, the catalase chemical equation was researched and deciphered, as the reactants and

products were studied as well. Next, the meaning of a substrate and a product was studied to then

formulate a solid definition. Last, we ran a standard test of the reaction between catalase and

H2O2 to precurse later experiments. Also, keep in mind, that all information obtained was

written down in the PLTW lab journal. In the standard experiment, we tested the efficiency of the

catalase enzyme in these standard conditions: the enzyme was added to 50 mL of 1.5% H2O2

with a pH of 6.5. Through lots of research it was discovered that the optimal pH for catalase to

operate is 7. So, the pH of the H2O2 in the standard reaction was brought as close to 7 as
possible. The standard experiment showed how effectively catalase operated at its optimal pH

with no pH buffer.

Hypothesis:

If the pH of the hydrogen peroxide solution is changed, then the closer the pH is to seven,

the more efficient the catalase enzyme will be, because the optimal pH for the catalase enzyme is

7.

Materials and Methods:

Materials:

Computer with Internet access and Vernier LoggerPro software

LabQuest Mini
Vernier Gas Pressure Sensor
Laboratory journal
Catalase solution, 400units/mL
50mL graduated cylinder
25mL graduated cylinder
Distilled water
125 mL Erlenmeyer flasks
Magnetic stirrer
Stirring bar
1.5% H2O2 solution
3% H2O2 solution
Ring stand
Utility clamp
pH meter
Two-hole rubber stopper assembly
Tubing with Luer-lock connectors
20-200 L micropippetor
200 L micropipette tips
25mL of pH 3 buffer
25mL of pH 5 buffer
25mL of pH 9 buffer
25mL of pH 11 buffer
Procedure:

1. LoggerPro software was started

2. The LabQuest Mini was connected to the computer using the USB cable
3. 50mL of 1.5% H2O2 solution was measured using the 50mL graduated cylinder into the

125mL Erlenmeyer flask

4. The stir bar was placed in the flask
5. The magnetic stirrer was placed on the base of the ring stand and a clamp was used to

fasten the flask to the ring stand. The flask was then positioned at the center of the

magnetic stirrer
6. The plastic tubing with two Luer-lock connectors was used to connect the two-hole

rubber stopper assembly to the Gas Pressure Sensor and the valve connected to the

stopper was closed

7. 100L of enzyme suspension was added to the contents of the flask using the

micropipette
8. The flask was tightly sealed by twisting in the two-hole stopper connected to the Gas

Pressure Sensor
9. The stirrer was turned on at a speed of 4
10. Data collection was started
11. After 200 seconds had passed, data collection was stopped
12. The stopper was removed from the flask and the contents of the flask were

disposed of in the sink

13. After the equipment was cleaned out, 25mL of pH 3 buffer were measured out in

the 25mL graduated cylinder and 25mL of the 3% H2O2 solution was measure out in the

50mL graduated cylinder

14. The pH buffer and 3% H2O2 solution were added to the 125mL Erlenmeyer flask
15. The stir bar was placed in the flask
16. The magnetic stirrer was placed on the base of the ring stand and a clamp was

used to fasten the flask to the ring stand. The flask was then positioned at the center of the

magnetic stirrer
 17. The plastic tubing with two Luer-lock connectors was used to connect the two-

hole rubber stopper assembly to the Gas Pressure Sensor and the valve connected to the

stopper was closed

18. 100L of enzyme suspension was added to the contents of the flask using the

micropipette
19. The flask was tightly sealed by twisting in the two-hole stopper connected to the

Gas Pressure Sensor

20. The stirrer was turned on at a speed of 4
21. Data collection was started
22. After 200 seconds had passed, data collection was stopped
23. The stopper was removed from the flask and the contents of the flask were

disposed of in the sink

24. The 25mL graduated cylinder and 125mL Erlenmeyer flask were carefully rinsed

out with water and cleaned

25. Steps 13-24 were repeated using the pH 5 buffer, pH 9 buffer, and pH 11 buffer

Results:

Each test was ran with a 1.5% H2O2 solution and was measured by noting the pressure

(kPa) per second (s) and comparing the slopes that was produced in each line. The first test result

that was obtained was the standard. This was performed with a 1.5% H2O2 solution and slope
that was made was .001737 kPa/s. The next tests were all performed by making a new solution

that changes the pH with pH buffers and still contains a 1.5% H2O2 concentration. The first test

had a 3.3 pH made with a 3 pH buffer and the slope that was made was a .01689 kPa/s. The

second solution that was tested had a 5.2 pH made with a 5 pH buffer and ended with a slope of .

02507 kPa/s. The third solution has a 7.8 pH made with a 9 pH buffer then produced a slope that

was .02890 kPa/s. The final solution consists of 8.1 pH made with a 11 buffer and had a slope

of .03380 kPa/s when the data was run.

Discussion:

Referring back to the background section, the optimal ph for catalase to operate in was 7.

The results refute to the information in the background, because it discussed how the optimal ph

for the catalase to operate in is 7, and the results say otherwise.

The results showed a steady increase from the phs 3.3 and 5.2, but then had a sudden

drop at the ph of 6.5, then went right back up to a gradual increase from the phs 7.8 and 8.1. The

solution in which the catalase was the most efficient was the ph level 8.1 with a slope of .03380.

The solution in which the catalase was the least efficient was the ph level 6.5 with a slope of .

001737. The graph showed the sudden decrease of the ph 6.5, this indicates that an error

occurred, such as the ph buffer did not change the 3% hydrogen peroxide back to 1.5% hydrogen

peroxide. If the ph 6.5 would have had normal results the graph would have a steady increased

line and ph level 3.3 would be the least efficient. Other possible errors in the experiment could be

that the two-hole rubber stopper was not completely covering the Erlenmeyer flask and the

maximum pressure was not reached. Another possible mistake that could have occurred is that

the dilutions were not correct or the lab group but too little of an amount of catalase enzyme.
 For improvement and elimination of any errors that occurred measure both parts of the

solution separately and double check the amount, to make sure the dilution will be effective. Also

test covering the Erlenmeyer flask with the two-hole stopper to make sure it fits and covers the

hole completely. Another improvement would be make sure to have the most updated version of

logger pro and a reliable device, also make sure to release ALL the catalase enzyme into the

flask. For further experiments, test more phs between, higher, and lower than 3,5,9, and 11, to

receive the most accurate results, then experiment with different dilutions of hydrogen peroxide

and the ph buffers for a full examination.

Conclusion:

In conclusion the hypothesis was not supported because generally the higher the ph

(excluding 6.5) the more efficient the catalase enzyme was.

Citations:

BBC - Standard Grade Bitesize Biology - Enzymes and aerobic respiration : Revision, Page 5.
(n.d.). Retrieved January 12, 2017, from
http://www.bbc.co.uk/bitesize/standard/biology/investigating_cells/enzymes_and_aerobic
_respiration/revision/5/
Introduction to Enzymes. (n.d.). Retrieved January 12, 2017, from http://www.worthington-
Biochem.com/introbiochem/effectsph.html
Williams, J. (1928, March 20). THE DECOMPOSITION OF HYDROGEN PEROXIDE BY
LIVER CATALASE. Retrieved January 12, 2017, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140981/