IB Biology Internal Assessment Higher Level (HL) on the effect of increasing copper (II) sulfate concentration on the rate of catalase activity in Solanum tuberosum, which received a score of 23/24 in the IB moderation, written by a student who received a 6 for the subject of Biology HL, and an ove...
Investigating the effect of inhibitors on catalase activity
As I live in a country with a diet rich in fish, I thought it would be interesting to investigate the
effect of a heavy metal inhibitor on catalase, since the amount of heavy metal ions in marine
environments has increased significantly over the past few years. I chose copper (II) sulfate as the non-
competitive inhibitor because heavy metals, such as Cu²⁺, are known to bioaccumulate markedly in
aquatic food chains. Also, as claimed by Solomon, copper is one of the most toxic metals to aquatic
organisms and ecosystems.1 With regards to crop production, Cu²⁺ contamination is a growing problem
all over the world due to human activities because this poses a soil stress to plant development (Ye et
al)2. Hence, this may affect plant catalase activity when present at toxic levels. Ultimately, potato
catalase was chosen over fish catalase as the latter wasn´t possible to obtain.
Enzymes are globular proteins that function as biological catalysts by reducing the activation
energy of the reactions that they catalyse. Catalase is a tetrameric enzyme, with each subunit being
formed by a single polypeptide chain with haemin as a prosthetic group (Vainshtein et al). 3Enzymes are
substrate-specific because the shapes of their active sites are complementary to the shape of substrate
molecules, thus allowing them to bind and fit together. When a substrate molecule successfully collides
with the active site of an enzyme, an enzyme-substrate complex is formed. Besides inhibitors, the other
factors affecting enzyme activity are temperature, pH and substrate concentration. All enzymes have
an optimum temperature and pH at which the rate of their activity achieves a peak. When the pH
increases or decreases from the optimum, the hydrogen ion concentration becomes lower or higher.
This alters the conformation of the active site of the enzyme and the charge of the protein. Below and
above a certain pH, acidity and alkalinity will denature the enzyme, which means that “the structure of
the enzyme is irreversibly altered” 4 and the substrate is no longer able to bind to the active site. If the
temperature is too low, the substrate and active site particles will not have sufficient kinetic energy to
collide successfully. However, if the temperature rises above the optimum, the bonds that maintain the
specific shape of the active site (e.g. hydrogen bonds) are broken and the chemical structure of the
active site is permanently destroyed (denaturation). As to substrate concentration, increasing it will only
increase enzyme activity up to a certain point (due to a greater frequency of substrate-active site
collisions), after that the active site will be saturated with substrate molecules and a plateau phase is
reached.
Enzyme inhibitors are chemical substances that reduce the activity of enzymes or prevent it
completely (Allot).5 A non-competitive inhibitor has a different shape to the substrate and so binds to
the allosteric site of the enzyme. This causes an irreversible conformational change in the shape of the
active site so that the substrate no longer fits perfectly into the active site; additionally, according to
Walpole, they may also partly block access of the substrate to the active site. 6 Subsequently, non-
competitive inhibition will not be overcome with increasing substrate concentration. On the other hand,
competitive inhibitors are chemically similar to the substrate molecule, so they occupy the active site
and prevent the substrate from binding7. As they are competing for the same active site, enzyme activity
will only increase when the inhibitor detaches or if substrate concentration increases.
In this investigation, I will be looking at how copper (II) sulfate, a non-competitive enzyme
inhibitor, affects catalase activity in Solanum tuberosum. Transition metal ions, such as Cu²⁺, are non-
competitive inhibitors to catalase because they are not structurally similar to the enzyme. According to
Yordanova, metal ions react with the iron in the prosthetic group of the enzyme. 8 Moreover, heavy metal
ions “form strong bonds with the carboxylate anions of the acidic amino acids or SH (sulfhydryl) groups
1
Solomon, F. (2009). Impacts of Copper on Aquatic Ecosystems and Human Health. [online] Ushydrotech.com. Available at:
http://www.ushydrotech.com/files/6714/1409/9604/Impacts_of_Copper_on_Aquatic_Ecosystems_and_human_Health.pdf [Accessed 25 Feb. 2020].
2
Ye, N., Li, H., Zhu, G., Liu, Y., Liu, R., Jing, Y., Peng, X. and Zhang, J., 2014. Copper Suppresses Abscisic Acid Catabolism And Catalase Activity, And Inhibits Seed
Germination Of Rice. [online] Academic.oup.com. Available at: <https://academic.oup.com/pcp/article/55/11/2008/2756072> [Accessed 15 October 2020].
3
Vainshtein, B., Melik-Adamyan, R., Barynin, V., Vagin, A. and Grebenko, A., 1981. Three-Dimensional Structure Of The Enzyme Catalase. [online]
https://www.nature.com/. Available at: <https://www.nature.com/articles/293411a0#citeas> [Accessed 25 February 2020].
4
Allott, A., & Mindorff, D. (2014). Biology: Oxford Ib diploma programme. p.99
5
Allott, A. (2014). Biology study guide. Oxford: Oxford University Press. p.100
6
Walpole,B (2016), Biology for the IB diploma, Cambridge University Press p.62
7
Allott, A. (2014). Biology study guide. Oxford: Oxford University Press. p.100
8
Yordanova, E. (1974). «IN VITRO» AND «IN VIVO» INHIBITORY EFFECT OF COPPER SULPHATE UPON THE ACTIVITY OF BLOOD CATALASE. [e-book] E.
Yordanova, p.p.98. Available at: <http://journals.mu-varna.bg/index.php/ssm/article/view/3553> [Accessed 21 March 2020].
1
, of cysteine, disrupting ionic bonds and disulfide linkages.” 9My research question is: What is the effect
of increasing copper (II) sulfate concentration (0.00 mol dm⁻³, 0.20 mol dm⁻³, 0.40 mol dm⁻³, 0.60
mol dm⁻³, 0.80 mol dm⁻³ and 1.00 mol dm⁻³), a non-competitive
inhibitor, on the rate of catalase activity in Solanum tuberosum, as
measured by the rate of pressure change (Pa s⁻¹) inside a sealed
container? As stated by Nandi et al., catalase is one of the most
important antioxidant enzymes. As it decomposes hydrogen peroxide to
innocuous products such as water and oxygen, catalase is used against
numerous oxidative stress-related diseases as a therapeutic
agent.”10Hydrogen peroxide is a major contributor to oxidative stress, as
it is converted to the hydroxyl free radical which leaks from the
mitochondria as it produces energy.11 I have a particular fascination for
the concept of oxidative stress, because it plays a major role in causing
chronic inflammation. This in turn has been shown by research to be a Figure 1- 3D structure of
possible root cause of many chronic diseases, such as heart disease. catalase (Nandi et al.)
2H₂O₂(aq)→ 2H₂O(l) + O₂(g)
Concentration of copper (II) sulfate solution (mol Rate of catalase activity in Solanum tuberosum
dm⁻³). The following range was chosen based as measured by the rate of pressure change
on preliminary trials. (due the production of O₂ in the decomposition
● 0.00 of H₂O₂) using a data logger with a pressure
● 0.20 sensor over 180 seconds inside a sealed
● 0.40 container (Pa s⁻¹).
● 0.60 Pressure change: ±2Pa
● 0.80 Time: ±0.01s
● 1.00
Volume of distilled water: ±0.05 cm³
Mass of copper (II) sulfate: ±0.001g
Table 1- Table to show the independent and dependent variable of this investigation.
Controlled variables:
● pH of reaction mixture
● Concentration and volume of H₂O₂ solution
● Mass of potato used
● Batch of hydrogen peroxide
● Sealed container properties
● Temperature
● Time
9
Chemistry LibreTexts. 2019. 2.5: Denaturation Of Proteins. [online] Available at:
<https://chem.libretexts.org/Courses/University_of_Arkansas_Little_Rock/CHEM_4320%2F%2F5320%3A_Biochemistry_1/02%3A__Protein_Structure/2.5%3A_Denatura
tion_of_proteins> [Accessed 7 May 2020].
10
Nandi, A. et al., 2019. Role of Catalase in Oxidative Stress- and Age-Associated Degenerative Diseases. Available at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885225/ [Accessed March 22, 2020].
11
ScienceDaily. 2009. How Oxidative Stress May Help Prolong Life. [online] Available at:
<https://www.sciencedaily.com/releases/2009/05/090528203726.htm#:~:text=This%20can%20result%20in%20significant,mitochondria%20as%20it%20produces%20ener
gy.> [Accessed 23 July 2020].
2
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