BIOLOGY 101 Laboratory 

A recurring topic in the study of biology is enzyme catalysis. There are over 2000 of these remarkable proteins already isolated and characterized. (Voet 1990) Most of an organism's metabolic activities are carried out by enzyme precision.

One of the by-products of many cellular reactions is hydrogen peroxide (H₂O₂). It is extremely toxic to living cells. There are some human immune system cells that actually use H₂O₂ to kill foreign invaders. Hydrogen peroxide is rendered harmless in cellular organelles called peroxisomes. Two examples of reactions that produce H₂O₂ are conversions of amino acids into "fuel" molecules and conversion of lipids to carbohydrates.

The question becomes, "if H₂O₂ is toxic and it is metabolically generated, what prevents cellular death?" In other words, what natural mechanism is in force to prevent H₂O₂ from killing a cell? Enter catalase. This enzyme is specific for H₂O₂ and it functions as follows:

All aerobic organisms use molecular oxygen, 3O₂, for respiration or oxidation of nutrients. During reduction of molecular oxygen to water, hydrogen peroxide is generated. It can damage DNA, protein and lipid membranes and may even be a causative factor in cancer. (Izawa, Inoue, Kimura 1996) For defense against H₂O₂, cells contain catalase. It is important to realize that each type of organism has its own catalase, which probably differs at least slightly in other species of organism. (Catalase Research Project 1998)

During this exercise, you will be investigating the following statements:

The rate at which enzymes catalyze reactions varies with their environment. Enzymes work best within an optimum range of physical and chemical conditions.

It is important to remember that enzymes act by binding substrates, forming a complex. The complex stresses chemical bonds forming a transition state. This makes the substrate more reactive. Energy is needed to form this state and it is provided by the enzyme. The enzyme's site of attachment and the parts that stress the substrate's bonds is known as the active site.

Some specific questions that you will investigate include: Will more enzyme complete the reaction faster? What will be the effect of substrate concentration on hydrolysis? What pH environment will enhance catalase reactions and which might retard them?

You may wish to see what the protein, catalase, looks like. The internet can be used to access Rasmol and a protein data base (PDB) file will allow you to access and view the enzyme in a 3-dimensional model. See appendix

A concept map for this Lab investigation is also found in the appendix

To do this experiment, six of the teams of students will carry out one version of the experiment using low, medium, and high enzyme concentration and a constant level of substrate, 1.0% H₂O₂. Then, the other six teams will do another version of the experiment using low, medium, and high H₂O₂ (substrate) concentration with cell extract diluted to 60% of its original concentration. At the end of the lab, experimental results will be pooled and the class as a whole will generate data showing the relationship between catalase activity and both enzyme and substrate concentrations.

A third experiment dealing with the effect of pH is provided. This portion could be performed easily by one or two teams of students.

MATERIALS AND METHODS (Per Team)

1. You will examine the effect of enzyme concentration on the rate of catalytic reaction. You have been given 10 mL of potato extract (catalase) which will be equivalent to your 100% solution. Set up dilutions of the catalase enzyme as shown in the table below. Complete the table for concentrations of 40%, 60%, 80% and 100%.

Example of calculation: You need 400 µl of volume and you want a 40 % solution of enzyme. 40% of 400 µl, is 160 µl. You then subtract 160 µl from 400 µl and this gives you the amount of buffer to be added to the solution. 

KEEP ALL ENZYME SOLUTION ON ICE THROUGHOUT THE EXPERIMENT

2. Obtain a flask of 1% hydrogen peroxide. This is the substrate for this part of the experiment.

3. Pipette 10 ml of 1% hydrogen peroxide into one of the small glass vials provided. Pick up a filter disk with clean forceps. Pipette 30 microliters of the 10% enzyme onto the filter paper disk. Avoid touching the disk with your fingers. Hold the disk for 5 seconds to ensure that the extract has been absorbed.

4. Your partner will time the reaction with a stop-watch or watch with a second hand. Place the filter disk containing the enzyme in the bottom of the glass vial containing the 1% H2O2. When the disk is at the bottom of the vial, immediately start timing.

5. Watch the disk carefully- you should be able to see bubbles appearing and eventually the disk will begin to rise. Stop timing when the disk reaches the top surface of the liquid. Record your results in your notebook in Table format.

6. Remove and discard the disk in a waste container. Repeat steps 4 through 6 two more times for the 10% enzyme concentration.

7. Repeat the above procedures for each of the enzyme concentrations. Note that you will have three sets of data for each concentration. Obtain an average time for each concentration. Plot 1/Rxn time (on Y axis) vs Enzyme Concentration in % (on X axis) Don't forget to title your graph.

1. Place 10 mls of your 0.3% hydrogen peroxide into each of 3 glass vials.
2. Pick up a filter disk with clean forceps. Pipette 30 microliters of a 60% enzyme concentration onto the filter paper disk. Remember your catalase stock is 100% and needs to be diluted. Avoid touching the disk with your fingers. Hold the disk for 5 seconds to ensure that the extract has been absorbed.
3. Your partner will time the reaction with a stop-watch or watch with a second hand. Place the filter disk containing the enzyme in the bottom of the glass vial containing the 0.3% H2O2. When the disk is at the bottom of the vial, immediately start timing.
4. Watch the disk carefully- you should be able to see bubbles appearing and eventually the disk will begin to rise. Stop timing when the disk reaches the top surface of the liquid. Record your results in your notebook in Table format.
5. Remove and discard the disk in a waste container. Repeat steps 4 through 6 two more times for the 0.3% substrate concentration. Why is it a good idea to do these tests in triplicate?
6. Repeat the above procedures for each of the substrate concentrations. Note that you will have three sets of data for each concentration. Obtain an average time for each concentration. Plot 1/Rxn time (on Y axis) vs Enzyme Concentration in % (on X axis) Don't forget to title your graph.

 

1. Obtain 5 test tubes and a test tube rack. Label the tubes pH 3-5-7-9-11.
2. Using a pipette and bulb or ratchet applicator, measure 4 mL of each of the pH solutions provided into the appropriately marked test tube. Be sure to replace the pipette in the appropriate pH solution. DO NOT MIX THEM OR LAY THEM DOWN.
3. To each of the test tubes, add 2 mLs of 60% catalase solution.
4. Swirl the test tube in order to mix the pH solution and catalase. Allow solution to sit for 5 minutes.
5. With a 1 mL pipette or a 1000 microliter micropipette and tip, measure 1 mL of 3% hydrogen peroxide into each of the test tubes. Allow 5 minutes for reaction time. Foam should form on the top of the solutions.
6. With your metric ruler, measure the distance from the bottom of the test tube to the top of the foam and record below.

1. Graph your results, using the X axis for pH and the Y axis for mm in height of solution/foam. Describe your results thoroughly and explain the effect of pH on enzyme activity.
2. Using the results of your pH investigation and your knowledge of the human digestive system, answer the following questions:

1. What is the approximate pH of the stomach?
2. What is the approximate pH of the beginning section of the small intestine.
3. Would the same enzymes which began the digestion of proteins in the stomach work as efficiently in the small intestines? Explain your answer.

 

Now that you have successfully completed a protocol for catalase analysis, choose another tissue to analyze as a mini research project. Suggestions might include: fruits, other vegetables, chicken parts or beef tissue. Does your model show more or less activity than a potato tuber? You may even decide to choose another potato type.

© 2000 Nancy L. Elwess

Last Modified: 06/15/00

By Sharon Clarke

E-mail: clar1838@mail.plattsburgh.edu