Observing the Effects of Concentration on Enzyme Activity

Ball-and-stick model of hydrogen peroxide

Hydrogen peroxide (H₂O₂) is a weak acid that is a colorless liquid at room temperature. Although it is primarily known for its usage as a solution in bleach, ripening agent, and disinfectant (National Center for Biotechnology Information, 2004), it is also known to be a chemical compound that is produced in the human body! One of the purposes of hydrogen peroxide is to fight off bacteria/infectious cells. When the immune system activates (in response to harmful bacteria that have invaded the body), mitochondria, in certain cells, produce hydrogen peroxide (Bao, Avshalumov, Patel, Lee, Miller, Chang, Rice, 2009) to eliminate the invading bacteria. Although helpful in keeping the body healthy, too much of H₂O₂ can cause health problems as deadly as cancer and diabetes (Wake Forest University Baptist Medical Center, 2008). To prevent the body from suffering the negative effects of hydrogen peroxide, an enzyme, known as "catalase", breaks down hydrogen peroxide into nontoxic substances.

Catalase is a protective enzyme found in the peroxisomes of nearly all anaerobic cells. Enzymes are a type of protein that speeds up chemical reactions that would otherwise be too slow at cellular temperatures. The haem-containing enzyme (Brugna, Tasse, Hederstedt, 2010) speeds up the breakdown of hydrogen peroxide into water and oxygen. If there were no catalase present in the human body, the human body would suffer the consequences of overflowing hydrogen peroxide. The chemical reactant speeded up by catalase is written as so: 2(H₂O₂) → 2H₂O + O₂ (arrow: catalase). Under conditions that are supportive towards catalase (exact pH, exact temperature), the catalytic rate for 1 molecule of catalase can break down 6 million molecules of hydrogen peroxide per minute (Prichett, 1998-2010)!

Catalase, a common enzyme found in organisms unprotected to oxygen.

Enzymes, as explained above, speed up chemical reactions. They take part in the reaction, but their shape is not changed by the reaction--they can be reused over and over again. They are also highly selective, only catalyzing specific reactions (this is because of their shape (Wake Forest University Baptist Medical Center, 2008)). Enzymes can denature if the pH changes or the temperature increases. This is because enzymes can only work at a specific pH and a specific temperature.

Researching the effects of concentration on enzyme activity is useful for people working in the medical field. This experiment can help figure out where enzymes are more efficient than others in the human body. If a person is diagnosed with cancer (which is believed to be influenced by hydrogen peroxide), health professionals, with background knowledge about where enzymes are more efficient, can put their skills to the test and prescribe certain medicines to help the enzymes eliminate the cancer cells in the body. As a future health professional, this lab can benefit me in my research as well.

This lab's purpose is to make use of a procedure that could be used to test enzyme activity in various body tissues. In this investigation, the different concentrations of catalase (100%, 75%, 50%, 25%, 0%) were the independent variable and the reaction rate was the dependent variable. The control group was the 0% catalase concentration. If 100% of catalase is applied, then the reaction rate will be maximum. This is because the more enzymes there are, the more sped up the reactions will be. Based on the ideas and research that we know about enzymes, it is believed that if 100% catalase is used, then the group receiving 100% will experience a maximized reaction rate.

Materials

1. Safety goggles
2. Medicine cups for dilutions (6)
3. Marking pencils
4. 100% catalase (stock) solution (30ml)
5. 10 ml graduated cylinder
6. Water
7. Stirring rod
8. Test-tubes
9. Dilute hydrogen peroxide solution (20ml)
10. Forceps
11. Filter paper disks (18)
12. Paper towels
13. Watch or clock with second hand
14. Metric ruler

Method

1. Put on safety goggles.
2. Determine how the experimenter will make a series of dilutions of the enzyme catalase. One way to make a dilution is to start with a 100% enzyme solution. To make 10 mL of a 50% enzyme solution, for example, mix 5 mL of water and 5 mL of the 100% enzyme solution. Complete a table showcasing how the experimenter will mix each of the enzyme dilutions needed in this lab.
3. Use a marking pencil to label 5 medicine cups for the enzyme solutions as follows: 100%, 75%, 50%, 25%, and 0%.
4. Obtain 30 mL of the 100% enzyme (catalase) solution.
5. Use a graduated cylinder to measure 10mL of the 100% enzyme solution into the medicine cup labeled "100%".
6. Prepare and label 10 mL of each of the dilutions according to the measurements in the enzyme dilution table. Mix each dilution thoroughly with a stirring rod. Note: Be sure to rinse the stirring rod with tap water after making each dilution.
7. Fill the test tube with 20mL of hydrogen peroxide solution. Note: If hydrogen peroxide solution is being measured in a graduated cylinder, clean the cylinder very carefully when finished. Mixing catalase and hydrogen peroxide is not desirable in this experiment.
8. 3 filter-paper disks must be placed into each cup of enzyme solutions. After 5 seconds, each disk must be removed and placed on a paper towel labeled with the dilutions so that they will not be mixed up until they are placed in the peroxide solution.
9. Using the forceps, place each disk into the hydrogen peroxide. Measure the time it takes for the disk to rise to the surface of the hydrogen peroxide. Begin timing as soon as the disk touches the surface of the hydrogen peroxide upon being placed in the peroxide. Use the metric ruler to measure the distance the disk sinks in the hydrogen peroxide. Multiply this measurement by two to determine the distance traveled. Enter the time and the distance traveled in the column for Trial 1 in the data table.
10. Repeat steps 8 - 11 for the 75%, 50%, 25% and 0% solutions. Complete the data table for each trial for each solution. Note: Be sure to use a clean filter-paper disk and a clean paper towel for each trial to avoid contamination.
11. Repeat steps 8 - 11 for the 0% solution. Note: If the disk has not risen to the surface within 3 minutes, write "no reaction" in the data table.
12. In Excel, plot a line graph of the reaction rates of the enzyme dilutions.
13. Dispose of solutions in the sink.
14. Clean up the lab area.
15. Hands must be washed after the lab.

Data

This data table presented here shows grouped raw data (in the form of a table) and the multigroup data average (at the bottom).

The group, "Connor, Adam, Brooke, Zoe", committed an error in their experiment with "0% Catalase", testing ".75%"--which differed with the rest of the other group's data average for "0% Catalase". Thus, their data was discarded (labeled "(discarded)").

These graphs represent the "Weston, Jeff, Aaqib, Emma" group's reaction rates (left) and the comparison between the "Weston, Jeff, Aaqib, Emma" group's average and the multigroup's average (right).

Results/Analysis

1. As proteins, enzymes contain peptide bonds. Describe a peptide bond.

   A peptide bond is a chemical bond created by the joining of the carboxyl group of a molecule and the amino group of another molecule along a protein chain. This releases H₂O.

2. What type of chemical reaction creates peptide bonds between amino acids?

   The type of chemical reaction that creates peptide bonds is dehydration synthesis/reaction.

3. Which concentration of catalase had the fastest reaction time, the slowest reaction time?

   The concentration of catalase that had the fastest reaction time was 75% catalase concentration, while the slowest reaction time was 0% catalase concentration.

4. Why did you measure the distance traveled by the disks to determine reaction rate?

   The distance was measured to determine the reaction rate because there had to be an accurate comparison between the different reaction rates for each disk.

5. Based on the graph and the overall slope of the line, what can you conclude about the effect of enzyme concentration on reaction rate?

   I can conclude about the effect of enzyme concentration on reaction rate that the more enzyme concentration, the more the reaction rate will be.

6. In the lab, the term 100% enzyme is the only relative - it is merely the concentration of the enzyme the teacher mixed. In other words, the enzyme concentration could have been much higher. Do you think that the trend noted in the graph above would continue if the enzyme samples were even more concentrated than those in the lab? Explain your answer.

   I think that the trend noted in the graph above would not continue if the enzyme samples were even more concentrated than those in the lab. If this were to be assumed, then the 100% enzyme concentration would have the highest reaction rate--but this was proven wrong. When data was being recorded in this experiment, the 75% enzyme concentration had a higher reaction rate than the 100% enzyme concentration. Similarly speaking, the 100% enzyme concentration could've had a higher reaction rate than 125%, 150%, 175%, and so on, concentration rates. Therefore, the trend noted in the graph would not continue if the enzyme samples were further concentrated.

Conclusion

In conclusion, the hypothesis ("If 100% of catalase is applied, then the reaction rate will be maximum") was not supported by my data. As noted in the data table, the column where the highest rate occurred is in the "75% catalase" column rather than the "100% catalase" column as my hypothesis stated. The reaction rate in the 100% catalase was 5.83 mm/sec while the reaction rate in the 75% catalase was 7.39 mm/sec. Therefore, the data recorded in the experiment does not support my hypothesis.

An error could've happened in several places in the experiment, such as when the dilutions were being mixed, the stirring rod could have been rinsed improperly, or never have been rinsed. This possibly would've altered the data that was being recorded and tracked. Other errors that would've changed the recorded data were mixing catalase and hydrogen peroxide in a graduated cylinder, incorrect timing, incorrect recording, etc.

References

1. National Center for Biotechnology Information. (2004). Hydrogen Peroxide. PubChem Compound Database. https://pubchem.ncbi.nlm.nih.gov/compound/hydrogen_peroxide#section=Top
2. Bao, L., Avshalumov, M. V., Patel, J. C., Lee, C. R., Miller, E.W., Chang, C.J., Rice, M.E. (2009). Mitochondria Are the Source of Hydrogen Peroxide for Dynamic Brain-Cell Signaling. Journal of Neuroscience 29 (28) 9002-9010. https://doi.org/10.1523/JNEUROSCI
3. Wake Forest Baptist Church. (2008). Hydrogen Peroxide Has A Complex Role In Cell Health. ScienceDaily. www.sciencedaily.com/releases/2008/01/080102134129.htm
4. Brugna, M., Tasse, L., Hederstedt, L. (2010). In vivo production of catalase containing haem analogs. Febs Press. Journal, 277: 2663–2672. http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2010.07677.x/full
5. Prichett, S. (1998-2010). How do enzymes work?. Inquiry Page. http://www.cii.illinois.edu/InquiryPage/bin/u12652.html