Introduction
The purpose of this procedure is to study the impact of various levels of salt in relation to the enzyme catalase during the breakdown of hydrogen peroxide. Catalase is important to the body because it breaks down hydrogen-peroxide from its toxic form (H202) into water and oxygen. Salt, or sodium-chloride, is a necessary electrolyte in maintaining homeostasis within the body, but its role with enzymes was unclear. Hence, to study the specific effect of salt on catalase in the process of hydrogen-peroxide decomposition, the amount of oxygen production was measured before and after the influence of various amounts of salt. In brief, an experiment was conducted, and showed the benefit of an optimum level of salt in the breakdown of hydrogen peroxide.
Hypothesis
If the amount of salt increases from 0 grams in the catalase solution, then less oxygen will be produced when the salt-catalase solution is mixed with hydrogen peroxide because the higher the amount of salt, the more it interferes with the function of catalase.
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Summary
This experiment tested the impact of salt on catalase activity in the breakdown of hydrogen peroxide. A filter paper soaked in a solution of salt and catalase was added into a vial of 20ml of 1.5% hydrogen-peroxide 7 times in two trials with varying levels of salt concentration. Salt levels were increased progressively from 0g-1.5g by .25g in the solution. In comparison to the enzyme activity in the absence of salt, the trials showed oxygen production peaking at 85-100mm/s with .75 grams of salt (fastest rise of filter paper). Hence, at .75g of salt, enzyme decomposition of hydrogen-peroxide was at its optimal point. By lessening salt levels by .25g-.5g from this point, oxygen liberation also decreased coherently. At this amount, the filter paper rose at only 30-60 mm/s. However, an increase of salt in the solution had similar effects on the release of oxygen. When salt levels were increased by .75g from the optimal point, the amount of reactions that occurred were significantly lower. At about 1.5g of salt within the solution, the smallest release of oxygen in any trial occurred. The filter paper rose at 32.6 mm/s.
Discussion
In a reaction with the enzyme catalase, hydrogen peroxide can easily break down, or decompose, into two parts: water and oxygen. By measuring the amount of oxygen liberation in a vial of hydrogen-peroxide, one can infer that this amount relates to the amount of reactions occurring between catalase and hydrogen peroxide. Therefore, the impact of salt on the enzyme’s activity can be observed through the variation in oxygen production.
After completing the experiment, research showed that there are very particular factors that affect the enzyme catalase’s sensitivity to salt. Specifically, when the enzyme is overexposed to salt, at 1-1.5g, the ions of salt denatures the enzyme in the secondary structure. This is due to the hydrogen bonds that make up the secondary structure of catalase, which are weak bonds that break easily if induced by another component in the environment. Consequently, the tertiary and quaternary structure of the enzyme are also altered, affecting its shape and function. In addition, too many ions causes an increase in the likelihood of collision between catalase and hydrogen-peroxide. This leads to protein unfolding. Protein unfolding creates changes in protein structure, affecting the enzyme’s ability to break down hydrogen peroxide.
Low concentrations of salt do not denature the shape of the catalase, yet they also do not influence catalase’s activity best. Although inorganic ions, like salt, may bind to some of the ionic side chains of catalase, it does not necessarily modify the three dimensional shape of the enzyme. As a result, the process of substrate molecules locating or binding to the active site of catalase can be faster with this amount of salt. Thus, the presence of any amount of ions from salt could alter the rate of the reaction. Even little amounts of salt (0.25-0.5 g), which contain inorganic ions help. In the case of the experiment measuring the breakdown of hydrogen peroxide and salt concentration, less oxygen would be produced when small amounts of salt are added, compared to .75 grams of salt. Low amounts of salt aren’t as effective as an optimum level of salt.
When there is the optimum amount of salt, the ions within the salt stabilize proteins by high-affinity binding to specific sites. This is a ligand-induced stabilization. A ligand-induced stabilization affects the shape or activity of a protein because ligands bind to a receptor of the enzyme. This increases the rate of the enzyme’s function.
An error in the experiment was within the effect of low amounts of salt in the solution on oxygen production. If there were no forms of human error, then a filter paper containing a low concentration of salt would have risen faster than a filter paper containing a high concentration of salt. This is because high amounts of salt cause catalase to become completely non-functioning after it is denatured by the salt. Fewer reactions can occur and only some oxygen can be produced. If there are low amounts of salt, however, then the shape of catalase is complete, and it can perform its function. This means that at low amounts of salt the rise of oxygen should be faster than when there are high amounts of salt. Yet, in our experiment, the difference in oxygen production within low and high amounts of salt are too similar. The cause of the error could simply be because the forceps used were hard to handle, and sometimes the experiment can be altered if one released the filter paper too early. Ultimately, the slight variation comes down to human error. Methods could be improved if students practiced using forceps.
In the human body, hydrogen peroxide is produced to protect from infection. Cells produce large amounts of hydrogen peroxide in response to the presence of bacteria. Hydrogen peroxide kills this bacteria. However, in higher amounts especially, hydrogen peroxide can be extremely toxic. As a result, catalase plays an important role in regulating amounts of hydrogen peroxide. In the liver, for example, the enzymatic activity of bovine liver catalase depends on the conditions of high pH and some ionic strength in an environment to decompose hydrogen-peroxide. Since salt supplies bovine liver catalase with an addition of ionic strength, its efficiency in performing its functions is increased. The liver fights infections by producing immune factors and removing bacteria and toxins from the bloodstream, with the help of catalase. However, what other environmental conditions contribute to the effectiveness of catalase? Where else can catalase be found in the human body? What are the effects of the digestion of hydrogen-peroxide?
Conclusion
The results did not prove the hypothesis. Instead of the linear relationship between an increase in salt and a decrease in oxygen production, the results showed that salt can support the function of catalase at an optimum level.
References
- Allison, T., M., Reading, E., Liko, I., Baldwin, A., J., Laganowsky, A., & Robinson, C., V. (2015, October 6). Quantifying the stabilizing effects of protein–ligand interactions in the gas phase. Retrieved from NCBI website: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600733/
- Kříž, Z., Klusák, J., Krištofíková, Z., & Koča, J. (2013, May 23). How Ionic Strength Affects the Conformational Behavior of Human and Rat Beta
- Amyloids – A Computational Study. Retrieved from Plos One website: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062914
- Lanyi, J., K, & Stevenson, J. (1969, January 23). Effect of Salts and Organic Solvents on the Activity of Halobacterium cutirubrum Catalase. Retrieved from JOURNAL OF BACTERIOLOGY website: https://jb.asm.org/content/jb/98/2/611.full.pdf
- Nishiura, J. (n.d.). Effect of salts and inorganic ions of enzyme activity. Retrieved from Brooklyn College City University of New York website:http://academic.brooklyn.cuny.edu/biology/bio4fv/page/salt_enz.htm
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