Abstract

Objective

The goal of this project is to investigate enzyme kinetics, using catalase enzyme extracted from potatoes. Enzyme activity will be measured as a function of temperature. A protocol for measuring activity as a function of enzyme concentration is also provided for those that have access to a 1 mL adjustable pipettor.

Introduction

One of the by-products of many cellular reactions is hydrogen peroxide (H₂O₂). It is extremely toxic to living cells. All aerobic organisms use oxygen for respiration or oxidation of nutrients. During reduction of molecular oxygen to water, hydrogen peroxide is generated. Two examples of reactions that produce H₂O₂ are conversions of amino acids into "fuel" molecules and conversion of lipids to carbohydrates. It can damage DNA, protein and lipid membranes and may even be a causative factor in cancer. There are some human immune system cells that actually use H₂O₂ to kill foreign invaders. The catalase enzyme is specific for the hydrolysis of H₂O₂:

Catalase is found in animal and plant tissues, and is especially abundant in plant storage organs such as potato tubers, corms, and in the fleshy parts of fruits. You will use catalase isolated from potato tubers and measure its rate of activity under different conditions.

Like other enzymes, catalase is a protein. Enzymes speed up chemical reactions by reducing the activation energy required to convert substrate(s) into product(s). Enzymes have specialized binding sites to do this.

Because enzymes are proteins, they are somewhat fragile. They can be denatured by heat, and can easily be broken down by proteases when cells are homogenized. To preserve activity of proteins in solution, it is important to keep the solutions on ice until you are ready to use them. Denaturing conditions, such as boiling, can also be used as evidence to show that an enzyme-based reaction is protein-dependent.

In the experimental protocol described here a filter paper disk will be immersed in a solution of the enzyme, then placed in the hydrogen peroxide. The oxygen produced from the subsequent reaction becomes trapped in the disc and will give it buoyancy. The time measured from the moment the disc touches the bottom of the container substrate to the time it reaches the surface of the solution is an indirect, but easily quantifiable measure of the rate of the enzyme activity.

Terms, Concepts, and Questions to Start Background Research

To do this project, you should do research that enables you to understand the following terms and concepts:

• enzyme,
• active site,
• substrate,
• catalase,
• anti-oxidants.

More advanced students will also want to study:

• activation energy,
• enzyme kinetics,
• Michaelis-Menten kinetics,
• K_m,
• V_max,
• double-reciprocal plot (also called a Lineweaver-Burk plot).
• What simplifying assumptions underlie the Michaelis-Menten analysis of enzyme kinetics? Are these conditions met in this experiment?

Questions

• What are the independent and dependent variables in this small experiment?
• How big is the variation between and within groups?
• How big is the variation within and between treatments?
• How does reaction rate vary with temperature? What causes this?
• How does enzyme activity vary with enzyme concentration? What causes this?

Bibliography

• Crook, J., 2003. "Catalase—An Extraordinary Enzyme," [accessed July 14, 2006] http://www.catalase.com/cataext.htm.
• SEPS, 200x. "Catalase FAQ," Science Education PartnershipS, Corvallis School District 509J, Oregon State University, and Hewlett Packard [accessed July 14, 2006] http://www.seps.org/faq/catalase.
• Wikipedia contributors, 2006a. "Michaelis-Menten Kinetics," Wikipedia, The Free Encyclopedia [accessed July 14, 2006] http://en.wikipedia.org/w/index.php?title=Michaelis-Menten_kinetics&oldid=62664791.
• Wikipedia contributors, 2006b. "Lineweaver-Burk Plot," Wikipedia, The Free Encyclopedia [accessed July 14, 2006] http://en.wikipedia.org/w/index.php?title=Lineweaver-Burk_plot&oldid=57071644.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• potatoes,
• gram balance, such as the Fast Weigh MS-500-BLK Digital Pocket Scale, 500 by 0.1 G available at Amazon.com
• blender,
• ice,
• insulated ice bucket or cooler,
• water baths at 0, 10, 30, and 40°C,
• 500 mL 1% H₂O₂,
• 1 L distilled water,
• 1 mL adjustable pipettor (e.g., Gilson PipetMan) and tips,
• filter paper disks,
• stop watch or watch with second hand,
• forceps,
• 5 50 mL beakers,
• 100 mL graduated cylinder,
• thermometer,
• 1.5 mL plastic microcentrifuge tubes.

Experimental Procedure

Extraction of Catalase

1. Peel a fresh potato tuber and cut the tissue into small cubes. Weigh out 50 g of potato cubes.
2. Place the potato cubes, 50 mL of cold distilled water, and a small amount of crushed ice in a blender.
3. Homogenize for 30 seconds at high speed. From this point on, the enzyme preparation must be carried out in an ice bath.
4. Filter the potato extract, then pour the filtrate into a 100 mL graduated cylinder.
5. Add ice-cold distilled water to bring up the final volume to 100 mL. Mix well. This extract is the 100% enzyme solution.
6. Note: This rough 100% enzyme solution should work OK although it is worth testing it before proceeding with the experiment. At room temperature (approx. 20°C) in a 1% H₂O₂ solution it would be sensible if the disk took about 20 seconds to rise in the beaker you are using. If it is faster than this, dilute the enzyme and use that as the 100% solution. If it is slower, prepare the extract again, starting with an increased amount of potato cubes. If the filter rises too quickly at room temperature, then the reaction at higher temperatures will be too quick to measure. If the filter rises too slowly, then the lower temperatures will take forever.
7. Keep your catalase preparation on ice.

The Effect of Temperature on Enzyme Activity

1. Label five 50 mL beakers with the temperature for testing (0, 10, 20, 30, and 40°C).
2. Add 40 mL of 1% hydrogen peroxide solution taken from the appropriate temperature water bath to each beaker.
3. Put the beakers in the appropriate water bath.
4. Using forceps, immerse a filter paper disk into the catalase solution you have prepared.
5. Allow the disc to absorb the enzyme solution for 5 seconds, then remove it and drain off the excess enzyme solution by touching the filter paper to the edge of the beaker.
6. Drop the disc into the first substrate solution.
7. The oxygen produced from the breakdown of the hydrogen peroxide by catalase becomes trapped in the fibres of the disc causing the disc to float to the surface of the solution.
8. The time (t) in seconds, from the second the disc touches the solution to the time it again reaches the surface is an indirect measure of enzyme activity.
9. Remove the disk from the beaker once it reaches the surface and dispose of it.
10. Record the time taken in a table in your notebook.
11. Clean the beaker and repeat the procedure until you have 5 replicates at the first temperature.
12. Repeat for each temperature so you have data for 0, 10, 20, 30, and 40°C.
13. Calculate mean and standard deviation for each temperature.
14. Construct and label a graph of your results.

The Effect of Enzyme Concentration on Reaction Rate

It is important to demonstrate that the enzyme assay shows that the enzyme actually follows accepted chemical principles. One way to demonstrate this is by determining the effect of enzyme concentration on the rate of activity while using a substrate concentration, in this case H₂O₂, that is in excess. This can be easily demonstrated with the experimental system used in the previous section. This set of experiments can be done most conveniently at room temperature.

1. Label 5 tubes 100%, 75%, 50%, 25%, and 0%.
2. Add 1, 0.75, 0.5, and 0.25 mL of 100% enzyme extract to the first 4 tubes .
3. Using a separate pipette, skip the first tube (100% enzyme) add 0.25, 0.5, 0.75, and 1 mL ice cold distilled water to the last four tubes and mix. Each tube will have now contain 1 mL of solution at the indicated enzyme concentration.
4. Keep all tubes on ice throughout.
5. Set up 5 beakers, each with 40 ml 1% H₂O₂ on the bench.
6. Check that the temperature in each one has equilibrated to room temperature.
7. Using the 100% enzyme solution, measure the reaction time using the disk enzyme assay for 5 replicate disks.
8. Repeat the procedure for each of the other four enzyme concentrations.
9. Plot the rate of reaction about enzyme concentration.

Variations

• Design and carry out an experiment to measure enzyme activity as a function of substrate concentration. Graph your results using a double-reciprocal plot.
• What happens to the enzyme activity if the catalase extract is subjected to conditions that denature proteins (e.g., boiling for 5 min)?
• For other Science Buddies projects involving enzymes, see: Liver Stinks!, Which Fruits Can Ruin Your Dessert? and A Juicy Project: Extracting Apple Juice with Pectinase.

• For more science project ideas in this area of science, see Biotechnology Techniques Project Ideas.

Credits

Sources

This project is from:

• Pritchard, J., 2002. "Teaching of Enzyme Biology Practicals," School of Biosciences, University of Birmingham, UK [accessed July 14, 2006] http://www.biosciences.bham.ac.uk/external/teachers/downloads/documents/handout.doc.

Edited by Andrew Olson, Ph.D., Science Buddies

Last edit date: 2011-11-15 09:00:00