Practical: The Effect of Different pH-Solutions on the Activity of Catalase

Hydrogen peroxide (H2O2) is a natural byproduct of the break down (and oxidation) of certain
molecules, such as fatty acids. Unfortunately, hydrogen peroxide is also toxic: it can destroy
cell membranes. In order to protect your cells, most reactions that produce hydrogen
peroxide occur in a cellular membrane-bound organelle called a peroxisome. If left on its
own, hydrogen peroxide will slowly decompose naturally into two harmless products, water
and oxygen.
2 H2O2→ 2 H2O + O2

Peroxisomes contain large amounts of the enzyme catalase, a protein that increases the
rate of hydrogen peroxide decomposition. Catalase ensures that hydrogen peroxide is
efficiently converted into water and oxygen, leaving your cells unharmed. This means that
the substrate of catalase is hydrogen peroxide. Catalase has a tremendously high turnover
rate: It is one of the fastest working enzymes and can decompose up to 40 million hydrogen-
peroxide molecules in one second, so that the poisonous hydrogen-peroxide can not harm
the cells. The cells in your liver contain many peroxisomes in order to effectively contain and
decompose the large amounts of hydrogen peroxide produced from the breakdown of
alcohol and other substances. Obviously, catalase is an important protein made by your
cells.
Catalase
2 H2O2 2 H2O + O2

This reaction produces O2 and also a lot of heat. So both could be measured to show the
activity of the enzyme in different pH solutions. You will measure temperature influenced by
the heat given off with a thermometer.

Our source of catalase is yeast, which contains the enzyme in sufficient concentrations.

Attention: Wear safety goggles all the time and handle hydrogen peroxide and the
buffer solutions with extraordinary care! These substances may cause
severe damage to eyes and skin!

Important: Note all your results for your lab report in a table.

Your report should include: 1. a focused research question

2. Introduction/scientific background

3. a hypothesis supported with scientific background (graph)

4. the identification of the relevant variables

and how they are controlled and measured

We will discuss how to write a quality lab report in the following lesson.
Material and Chemicals (for one student/one group):

• Buffer solutions of the following pH values: 3, 5, 7, 9, 11 (10 ml of each pH buffer). The buffer
solutions and the respective pipettes are on the teacher’s desk.
• Yeast catalase (yeast contains the enzyme catalase; concentration of yeast: 10 g in 100 ml of
water = 10% yeast solution; the solution will be provided the teacher)
• 15% H2O2 (5 X 10 ml = 50 ml)
• 1 x 400 ml beaker (small and high shape)
• 1 x 10 ml measuring cylinder
• 1 small funnel (for the measuring cylinder)
• 5 x 10 ml pipettes and pipette teats (to pipette the pH buffers into the plastic cups). The buffer
solutions and the pipettes are on the teacher´s desk
• 1 x 2ml syringes
• 1 plastic cup
• Magnet stirrer with magnet and 150 ml beaker containing the enzyme solution (for all students
on the teacher’s desk)
• Thermometer
• Stop-watch
• Safety goggles

Method:

1. Put the plastic cup into the 400 ml beaker.

2. Add 10 ml of pH7 buffer and 10 ml of the 15% H2O2 solution to the plastic cup.
• For the buffer use the pipette and pipette teats.
• For the H2O2 solution put the funnel into the graduated cylinder to measure 10 ml.
3. Put the thermometer into the solution and measure ~after 1 min the starting temperature of
the solution for the following experiment.
4. Add 1 ml of the yeast catalase solution into the pH/H2O2 solution with the help of a syringe.
Start the stopwatch and simultaneously stir the solution shortly with the thermometer.
5. Measure and the temperature every 30 s for 5 min. Write the numbers down.
6. Pour the solution of the plastic cup into the provided waste tank for chemicals and rinse and
clean the plastic cup and the thermometer
7. Repeat steps 1 – 6 for the other pH values.
8. Clean the apparatus and put everything back to the teacher’s roller table.

Some advice for a good lab report:

• Do not forget to note associated qualitative data during the experiment!

• To get a very good grade in the analysis part, statistical analysis is required. So you should
think about getting raw data from other groups during the experimental lesson already.
• Do not forget to add this instruction sheet to your report (then you do not have to write the
method and the materials list on your own)

Good Luck!