Objective

To investigate the effect of copper sulphate and concentration of hydrogen peroxide solution on
catalase activity in yeast cells

Biological Principle
Enzymes
Catalase is an enzyme.
Enzymes are biological catalysts. They lower the activation energy for a reaction by binding to
the substrate, making the reaction more easy to take place. Thus speeding up the rate of a
reaction.
Enzymes are protein in nature. Amino acids are joined together by peptide bonds to form
polypeptides, which form proteins. The attractive force between different amino acids causes
the polypeptide chains to coil and fold in specific ways to form a 3D structure.
Enzymes have active sites that are specific in shape. Hence they are specific in action as only
substrates that can fit into their active sites can bind to the enzyme to form an enzyme-substrate
complex for the enzyme to catalyse its reactions.
Enzymes are sensitive to temperature and pH. At low temperatures, enzymes are inactive. The
substrate molecule and enzyme molecule have lower kinetic energy so they move at slower
speeds. The chance of collision between substrate molecules and enzyme molecules are lower.
Hence the chance of forming enzyme-substrate complexes are lower. As a result, less reactions
are catalysed by enzymes and the rate of reaction is lower. At high temperatures, enzymes
become denatured. High temperatures cause the active site of enzymes to change shape.
Substrates cannot fit into the active site and bind to the enzyme. No enzyme-substrate complex
can be formed. Enzymes lose their catalytic ability permanently. (It cannot be restored even if
cooled down to lower temperatures). At unsuitable pH, the active site of enzymes also changes
shape and the enzyme becomes denatured, losing its ability to catalyse reactions.

Hydrogen peroxide
Catalase catalyzes the breakdown of hydrogen peroxide into oxygen and water.

Copper sulphate
Copper(II) ions are heavy metals. It is an inhibitor of catalase by binding to catalase’s active site.
Substrates cannot bind to catalase’s active site to form enzyme-substrate complexes. Catalase
cannot catalyse the breakdown of hydrogen peroxide. Hence, enzymatic activity is inhibited.

Design of Experiment
Variables
IV
 1) Concentration of hydrogen peroxide solution
2) Presence of Copper sulphate solution
DV Time needed for yeast bead to raise to the surface
CV
1) Volume of hydrogen peroxide solution in each trial
2) Mass, volume (density) and shape of each yeast bead
3) ???

Design of Experiment
Brief description
In this experiment, 3 plastic cups will be filled with an equal amount of hydrogen peroxide
solution with different concentrations (1.5%, 1.5%, 3%) and label them A,B,C respectively.
5 yeast beads will be dropped into each cup one at a time. The yeast beads dropped into A
should be pre-immersed in copper sulphate solution. The time for each yeast bead to raise to
the surface will be recorded. HIII

Prediction
As concentration of hydrogen peroxide increases, it is predicted that catalase activity increases.
The concentration gradient of hydrogen peroxide in the solution and cytoplasm of yeast cells
becomes steeper. Thus, the diffusion rate of hydrogen peroxide into the yeast cells increase.
The chance of collision between catalase in yeast cells and hydrogen peroxide molecules
becomes higher and so is the chance of forming enzyme-substrate complexes. More
breakdown of hydrogen peroxide is catalyzed by catalase. The reaction rate will be higher. Rate
of production of oxygen is higher. Less time is required for yeast bead to raise to the surface.

With the addition of copper sulphate solution, it is predicted that catalase activity will decrease.
Copper(II) ions diffuse into the cytoplasm of yeast cells across its cell membrane and bind to the
active site of catalases. It prevents hydrogen peroxide from binding to catalase to form
enzyme-substrate complexes and thus lowers the rate of reaction catalyzed by catalases. Rate
of production of oxygen is lower. More time is required for yeast bead to raise to the surface.

Procedures
Part 1: Preparation of Yeast Beads
1. Prepare a 10% solution of dried yeast
2. Add aliquots (2 cm3) of the stock yeast solution to a solution
of sodium alginate (2%, 2 cm3) and mix thoroughly.
3. Place the yeast/alginate mixture into a syringe positioned
above a solution of 2% CaCl2 (Figure 1).
4. Allow the liquid to flow;
5. the drops form balls of immobilised yeast that are left in the
CaCl2 solution for about 5 minutes
6. Washed the yeast beads gently under running cold water
7. Rinse the yeast beads with distilled water.
Part 2: Effect of concentration of hydrogen peroxide solution on Catalase Activity
1. Add 10mL of 3% hydrogen peroxide solution to 3 graduated cylinders each.
2. Label them ABC
3. For cylinder A and B, add 10mL of distilled water to dilute the hydrogen peroxide solution
to 1.5% concentration; For C, add another 10mL 3% hydrogen peroxide solution.
4. Use forceps to drop one yeast sphere into the graduated cylinder A.
5. Start timing as soon as the sphere touches the bottom of the cylinder.
6. Stop the timer when the sphere reaches the surface.
7. Record the time needed for yeast bead to raise to the surface
8. Repeat step 4 to 6 using 5 new yeast beads for cylinder A.
9. Repeat the step 1 to 8 for cylinder B and C respectively.

Part 3: Effect of copper sulphate solution on Catalase activity

1. Immerse 5 yeast beads in the copper sulphate solution for 5 minutes
2. Add yeast beads to hydrogen peroxide solution

Precautions
1. Immerse the yeast beads in copper sulphate solution for 5 minutes before adding to
hydrogen peroxide solution
2. The yeast bead, copper sulphate solution and hydrogen peroxide solution should have
the same temperature before starting the experiment.
3. Wear gloves when handling H2O2 solution as it is highly corrosive
4.

Results

Concentration Time needed for yeast bead to raise to Enzymatic Rate (s-1)
of hydrogen the surface （s）
peroxide
solution (%) Trial 1 Trial 2 Trial 3 Average

0.75 7 7 7 7 0.143

1.5 4 5 6 5 0.2

3 3 1 2 2 0.5

Treatment of Copper Time needed for bead to raise to the surface (s) Enzymatic
Sulphate solution to yeast Rate (s-1)
 beads Trial 1 Trial 2 Trial 3 Average

Without being treated with 5 6 4 5 0.2

Copper sulphate solution

Being immersed in copper 16 15 17 16 0.0625

sulphate solution

Graph 1: H2O2 - line graph

Title of Graph: Enzymatic rate of Catalase in Yeast Cells calculated by time needed for yeast
bead to raise to the surface in different concentrations of hydrogen peroxide

Graoh 2: CuSO4 - Bar Chart

Title: Enzymatic rate of Catalase in Yeast Cells calculated by time needed for yeast bead to
raise to the surface …?

Discussions
- Higher or Lower catalase activity after treatment of CuSO4
- Slope of increase / levels off →

Sources of error, limitations & improvements

Limitations/Errors Improvements

Human error in time recording &
judgement of whether yeast bead
raise to the surface is subjective
Use hydrogen peroxide solution of lower
concentration. The rate of breakdown of hydrogen
peroxide is lower and the rate of release of oxygen is
also lower. The time needed for the yeast bead to
travel to the solution's surface is longer. Percentage
error due to human reaction time is lowered,
increasing the accuracy of the result

1. The number of yeast cells in Do more trials and take an average of replicates →
each yeast bead is different reduce error due to individual differences in yeast
2. Catalase content in each bead → minimize sampling error
yeast cell is different

The volume and mass of each yeast We can use a micropipette to replace the syringe as it
bead is different can measure volume more precisely, which can
provide a more accurate result → improve the
accuracy
 get a relatively more constant volume of yeast to make
the yeast bead

Conclusion
When the concentration of hydrogen peroxide solution increases from __% to __ %, the
catalase activity increases/
The higher the concentration of hydrogen peroxide solution, the higher the catalase activity

Copper (II) ions (, which are inhibitors of catalase,) lower or stop the activity of catalase.