The Effect of pH on Catalase Activity

Research Question
How does the pH environment (pH 4,6,8,10 and no buffer as a control) of the enzyme catalase (taken from the liver of Ovis aries) affect the rate at which Hydrogen Peroxide (H2O2) is broken down (measured in seconds, with ± 1s error)?
Personal Engagement
Ever since reading a fascinating article[1] on the importance of catalase to the process of life, I have been intensely captivated by the process by which food is digested. My interest was especially piqued when I discovered it helped to break down the toxic hydrogen peroxide in our bodies – but that this hydrogen peroxide is only produced in the first place to prevent the formation of a more toxic substance – superoxide, which ‘rips ions apart’[2]. I was also interested to learn that catalase has the highest turnover numbers of all enzymes – making it one of the fastest acting enzymes in existence.
Design of Experiment
I was heavily involved in a personal capacity with the design of the experiment. This was achieved through the conduction of a pilot study, where I chose to investigate the effect of pH on the activity of catalase (instead of the effect of changing temperature – an option which was also thought about).
In the pilot study, there was a range of independent variables, including the catalase concentration, the volumes of the substances, the volume of the pH buffer and the concentration of hydrogen peroxide After a good deal of trial and error, I decided to use 40 ml of 5vol concentration hydrogen peroxide, and a separate beaker containing 50ml of catalase solution – at a 0.1% concentration. After deliberation, it was decided that the pH buffer (the independent variable) was to be tested at graduated intervals of pH 4, 6, no pH (to serve as a control – leaving the pH at 7), 8 and 12. 15ml of each pH buffer was to be added each time, since during the pilot study we discovered that using 10ml did not give as pronounced effects as desired.
Background to Enzyme Catalysis
Enzyme catalysis requires that the substrate be brought into close proximity with the active site. When a substrate binds to the enzyme’s active site, it forms an enzyme-substrate complex and the enzyme catalyzes the conversion of the substrate into product (this is the ‘chemical reaction’), creating something called an enzyme-product complex. The enzyme and product then dissociate and since the enzyme was not consumed or used up in the process, it can continue to catalyze further reactions.

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Hydrogen Peroxide (H2O2) is a toxic substance in the human body, which is often said to be created ‘by accident’ in respiration. The catalase enzyme breaks it down into Hydrogen (H2) and Oxygen (O2). This is an example of the liver performing its function of using specialized enzymes to help it break down toxic substances and thus make them safer for the body to process.
Hypothesis
The prediction made at the start of the experiment was that the optimum pH for catalase activity was to be somewhere between pH 6 and 8. In the context of the experiment the prediction was that between pH 6 and 8, the time taken for the paper disc to rise would be lowest on average (due to faster H2O2 decomposition). At extremely high pH levels, the charge of the enzyme will be altered. This changes protein solubility and overall shape. This change in shape of the active site diminishes its ability to bind to the substrate, thus annulling the function of the enzyme (catalase in this case). This process of denaturing only occurs when the enzyme is operating in an environment outside of its optimum range. It is hypothesised that the optimum range for catalase in this IA is between pH 6 and pH 8.
Independent Variable
The independent variable is the value of the pH buffer (from 1-7) which was used in each beaker of H2O2. 15ml was added each time using a graduated syringe.
Dependent Variable
The dependent variable is the time taken for the paper disc to fall through the 40ml of hydrogen peroxide and rise to the surface of the beaker. It was measured in seconds (s), with an error allowance of ± 1 second, to account for human error on the stopwatch.
Control Variables  
There are four main control variables, which needed to be accounted for in this investigation. Temperature, substrate concentration, enzyme concentration and the presence of an inhibitor all needed to be controlled in order to produce valid results.
Temperature 
If the temperature is low, then this can result in there being insufficient thermal energy to meet the activation energy of the reaction – the decomposition of hydrogen peroxide by catalase. If the temperature is increased then the higher kinetic energy will result in more successful collisions between catalase enzymes and hydrogen peroxide substrates. At the optimal temperature – which is 37°C for catalase, then the rate of enzyme activity will be at its peak. Both high and low temperatures (approximately 10°C either side of the optimum) will cause enzyme stability to decrease – since the change in thermal energy will disrupt the hydrogen bonds within the enzyme. This can cause denaturation, following the loss of shape of the active site.
How is it controlled?
The entire experiment was conducted in a lab – which had a constant temperature of 25°C. In order to increase the temperature of the catalase to the required ‘optimum’ level, a water bath – measured electronically to be 37°C was used. This was monitored throughout the experiment to avoid any unwanted fluctuations. Windows were also covered with blinds to prevent any increase in the general temperature of the laboratory as a result of the sunny conditions outside.
Substrate Concentration and Enzyme Concentration
For similar reasons to temperature, increasing the substrate concentration (concentration of hydrogen peroxide) or the enzyme concentration (catalase) (initially, will lead to an increase in the rate of reaction. This is because there will be a greater number of successful collisions per unit time between substrate and enzyme. However, beyond the optimum level of substrate concentration – which through our pilot study we found to be 5vol. – the solution becomes saturated, and there is no further increase in rate. Similarly, saturation occurs beyond the optimum level of enzyme concentration – which we found in our pilot study to be 0.1% catalase.
How is it controlled?
Care was taken to ensure that the correctly labelled solution of hydrogen peroxide was used in each of the five beakers at each of the five pH levels. It was replaced every time the pH was changed (i.e. five times). Care was also taken to ensure that the paper disc was not mistakenly dipped into a stronger or weaker beaker of catalase than the 0.1% concentration that was decided upon.
Presence of Inhibitor
Inhibitors essentially alter the catalytic action of the enzyme – and can slow down and even stop the process of catalysis. This can be done by competitive, non-competitive and substrate inhibitors. In the case of catalase, two non-competitive inhibitors were identified for special concern. Any heavy metal ions (such as CuSO4) and also potassium cyanide (KCN) can bind to the catalase and decrease its activity.
How is it controlled?
Before the experiment began, all surfaces, beakers and other apparatus were sterilized and thoroughly cleaned, in order to create an environment that did not contain any enzyme inhibitors.
Thus the variables in the environment were kept constant (controlled) to allow for valid results by changing a single variable in the experiment – pH.
Method
A pilot study was undertaken so that the most effective values – concentrations and volumes – could be determined ahead of the full investigation. In order to represent the full range of the pH scale, values of 4, 6, 8 and 10 (and no pH – leaving the pH at 7 for a control) were used. The time taken for the paper disc to rise and fall was too short when the 5% catalase was used, and similar problems were encountered when a concentration greater than 5vol. was used for the hydrogen peroxide. Finally, due to fluctuation in the results, it was decided to repeat the experiment 5 times at each pH, instead of the original 3 times. It was hoped that this would increase reliability.

Pour 40ml of Hydrogen Peroxide (H2O2) at 5vol. concentration into five 50ml beakers.
Pour 50ml of catalase solution at 0.1% concentration into a large beaker – for dipping the paper discs.
For four of the beakers, add 15ml of the selected pH buffer (beaker 1 – pH 4, beaker 2 – pH 6 and so forth all the way to pH 10). The fifth set should be left without a pH buffer to act as a control.
This 15ml should be measured carefully using a graduated syringe.
Using the forceps, immerse a paper disc of regular and consistent shape and size (taken from a collection of hole punch detritus) into the catalase solution. Ensure it is well mixed.
Remove and shake off any clearly excess catalase solution from the disc.
Drop the disc into the beaker of hydrogen peroxide from the consistent height of the top of the beaker, and start the stopwatch as soon as the disc hits the surface of the solution for the first time. Remove and discard the disc into a pre-prepared waste beaker.
If the disc settles at the side of the beaker or gest caught on the sides, remove it with the forceps and repeat that trial.
Note that the hydrogen peroxide only needs replacing when the pH is altered and should not be replaced between repeats at the same pH value

Equipment List – Apparatus and Chemicals

Hydrogen Peroxide (5vol.) – 200ml
Measuring cylinders – 2 x 50ml (± 0,05ml)
Beakers – 5 x 50ml (± 0,05ml)
Syringes – 5 x 15ml (± 0,05ml)
Stopwatch – the iPhone app was used since it was deemed easier to use than the analogue stopwatches provided by the school (± 1 second)
Catalase source from liver of Ovis aries – 50ml at a concentration of 1% in 1 litre of water
pH buffer – 15ml each of pH 4, pH 6, pH 8 and pH 10
Paper discs – 25 (plus ample spare discs in case of mistakes)
Forceps – thoroughly cleaned beforehand

Risk Analysis
Safety goggles were worn throughout the experiment due to the fact that most enzymes are sensitizers, and could potentially cause breathing difficulties if inhaled. Additionally, many enzymes – such as catalase can irritate the eyes and the skin. However, due the fact that the catalase enzyme solution used was concentrated at less than 1%, it was deemed unlikely that the experiment contained any significant risk to the group
Ethical Considerations
On the whole, there were no ethical considerations in the actual practice of the experiment. However, during the pilot study, the group expressed concern over the potential exploitation of the Ovis aries subject from which the liver catalase was obtained. However our teacher assured us that the lamb used had been housed in the most ethically aware institution in Britain, and that the catalase had been obtained without causing pain to the animal.
Results
Table of Raw Data to show the time taken for the paper disc to rise at different pHs and thus the level of catalase activity at different pHs

pH (1-7)

Time Taken (s) (±1s)

Time Taken (s) (±1s)

Time Taken (s) (±1s)

Time Taken (s) (±1s)

Time Taken (s) (±1s)

 

Trial 1

Trial 2

Trial 3

Trial 4

Trial 5

No

2.86

4.50

4.2

6.5

4.48

4

8.25

7.98

10.13

9.76

8.53

6

6.82

6.65

6.50

6.10

5.56

8

6.18

5.55

5.52

6.22

5.56

10

6.76

6.89

7.48

5.78

6.83

Table of Processed Data to show the time taken for the paper disc to rise at different pHs and thus the level of catalase activity at different pHs

pH (1-7)

Mean Time Taken (s) (±1s)

Standard Deviation

No

4.51

1.16

4

8.93

0.855

6

6.33

0.504

8

5.81

0.322

10

6.75

0.548

A mean was calculated in order to incorporate all of the data values collected and to counteract the detrimental effect of anomalies. This was done using the following formula.
The mean transmission for temperatures at pH 6 for example:
              (6.82 + 6.65 + 6.50 + 6.10 + 5.56) ÷ 5 = 6.33 seconds (± 1s)
The standard deviation was calculated to show how much the results varied from the mean on average (the spread). This was done using the following formula.
The standard deviation for pH at pH 6 for example:
The mean is equal to 6.726 and the sum of all deviations from the mean is 1.0172.
Hence standard deviation can be worked out by solving the following equation:
σ=∑(Xi– Χ̅)2n–1= 1.01725–1
= 0.504
Error bar = 1 standard deviation which is different for each data point
There were no especially anomalous results exhibited
Conclusion
 The trend of the data obtained overwhelmingly supports the hypothesis that was made. It is clear that as pH increases and decreases from pH 7, then the time taken for the paper disk to rise and fall through the hydrogen peroxide solution increases. This shows that the more pH varies from the optimum level of pH7, the less active the enzyme catalase becomes in breaking down the hydrogen peroxide, because at low and high pHs less gas is being produced at the same rate to propel the disc upwards.
For example, the two most extreme pH values at which the experiment was conducted (pH 4 and pH 10) were also the values at which the paper disc took the most time to fall and rise – 8.93s and 6.75s respectively. This contrasts directly to the amount of time taken by the disc at the optimum pH of 7 – where the average value was the much lower 4.51s. This follows the trend observed in most scientific investigations about the effect of pH on enzyme activity – and resembles the hypothesis diagram from the start of this report. The conclusion to draw from this is that enzymes require a very specific pH in order to operate most efficiently.
The results do not entirely represent the perfect trend which was hypothesized at the beginning though. It was expected that the mean time taken (s) (± 1s) for pH 4 and pH 10 would be more similar than 8.93s and 6.75s respectively, due to the fact that these two values are the same difference from the optimum pH (of 7). One possible reason for this is that the amino acids which make up the catalase enzymes are more resistant to alkaline than acidic conditions (since the time taken was longer at the lower pH) – indeed there is a good deal evidence in scientific literature[3] that this is the case. However, the conditions in the human liver for catalase enzyme activity occur at pH 7, suggesting that due to adaptation over time, the body has determined that a neutral pH provides the very best conditions for catalase to operate under.
On the whole though, in this investigation pH clearly has had an effect on the ionization of amino acids – the proteins that make up the catalase enzyme. Acidic amino acids contain carboxyl functional groups in their side chains. Basic amino acids contain amine functional groups in their side chains. The state of ionization of the amino acids is altered when the pH is changed. If the conditions become more acidic, then due to the proliferation of H+ ions, the charge becomes more positive. If the conditions become more alkaline, then due to the proliferation of OH– ions, the charge becomes more negative. As a consequence of this, the hydrogen bonds that determine the 3-D shape of the protein are altered. This is known as denaturing and causes a diminishing activity level from the enzyme catalase. Changes in pH do not only affect the shape of the catalase enzyme but also change the shape or charge properties of the hydrogen peroxide substrate so that the hydrogen peroxide will not bind to the active site and thus cannot undergo catalysis.
Evaluation – Data Reliability
On the whole, the data that was obtained exhibited a good level of reliability. The standard deviation was low for all data points, with the maximum being 1.16. Hence the data was clustered around the mean, illustrating the fact that there were no wild variations. This consistency is perhaps down to the fact that following the pilot study, where we found the stopwatch quite difficult to use accurately, the iPhone app was used instead – something which enabled us to be far more precise and to obtain more reliable results. It is perhaps down to this that there were no anomalies. The number of data repeats – increased to 5 from an initial 3 in the pilot study – was perfect, both in terms of timing – which emerged as a logistical concern, but also in ensuring that a representative average could be obtained from the data.
Evaluation – Limitations and Improvements

Limitation of Experiment

Systematic (method) or Random (experimental errors)

How could this have affected the results and hence the conclusion?

Suggestion for amelioration of this problem

Beaker size not entirely consistent – since they are designed by humans, and due to meniscus effect the volume of substance in the beaker may be deceptive

Systematic

This created room for error with regards to the measurements – if too much hydrogen peroxide had been used then the effect of the pH changes on the experiment would be less pronounced and would thus lead to less clear results. This is due to the fact that the human eye is not precise in analysing such measurements

Spend some more of the science department budget on mathematically produced beakers which are all precisely the same size. The meniscus effect in liquids can be overcome by using a goniometer – which is an instrument that measures contact angles

Paper Discs sank inconsistently

Systematic

Sometimes the catalase in the filter paper disk reacted too quickly/not quickly enough with the hydrogen peroxide, since there had been different levels of absorption into the paper. This may have contributed to results being variable, as due to this flaw in the method it is difficult to standardise the results with the same conditions

A different measurement technique for the production of gas could be used – perhaps a gas syringe if the experiment was conducted in a different way, using different volumes and concentrations of the enzyme and the substrate

Catalase concentration varied wildly due to imprecise source (could have come from all sorts of different Ovis aries whose liver concentration of catalase would fluctuate greatly) – so difficult to be exact

Systematic

Since the higher the concentration of catalase, the higher the rate of reaction, this variation of the nature of the catalase used in different parts of the lab could have caused a lack of accuracy between the sets of trials at each pH value

In the future if catalase was taken from the livers of Ovis aries that exhibit similar physical characteristics (size, age, gender etc.) or even simply from the same animal this would improve the accuracy of the results since the catalase concentration would be more consistent. The logistical and financial difficulties of this solution are, of course, noted.

Stopwatch inconsistencies remained as a result of human reaction times – despite switching to a more precise method

Random

This would have directly affected the results – since time was being measured as the dependent variable.

Perhaps a more sophisticated or detailed system of time measurement could be used – such as milliseconds. However this does not solve the inherent (and perhaps unsolvable) problem of the delay in human reaction times. Maybe a robotic solution is the only way to eliminate this, with sensors of some sort automatically timing the journey of the paper disk as it falls and rises through the hydrogen peroxide.

Ranking of Limitations (1 = most important/significant)

Stopwatch inconsistencies – because it is the one that directly influences the final results in the largest way, given that the average human delay in reaction time is approximately 0.25 seconds for a visual stimulus[4].
Paper Disc inconsistencies
Beaker Size inconsistencies
Catalase Concentration inconsistencies

Bibliography – All Accessed May 2019
–          http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm
–          https://namedprogram.com/understanding-alkalinity-and-acidity-version-2-0/
–          https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Physical_Properties_of_Matter/States_of_Matter/Properties_of_Liquids/Contact_Angles
–          https://backyardbrains.com/experiments/reactiontime
–          https://sciencing.com/ph-levels-catalase-6826245.html
–          https://www.scientificamerican.com/article/exploring-enzymes/
–          https://www.mcgill.ca/oss/article/general-science-you-asked/hydrogen-peroxide-bodys-best-defence-system

[1] Article about enzymes https://www.scientificamerican.com/article/exploring-enzymes/ (accessed May 2019)
[2] Article about superoxide https://www.mcgill.ca/oss/article/general-science-you-asked/hydrogen-peroxide-bodys-best-defence-system (accessed May 2019)
[3] Optimum pH conditions for catalase activity are said to be between pH 7 and pH 11 – https://sciencing.com/ph-levels-catalase-6826245.html (accessed May 2019)
[4] ‘The average reaction time for humans is 0.25 seconds to a visual stimulus’ – https://backyardbrains.com/experiments/reactiontime (accessed May 2019)