THE EFFECTS OF VARYING CONDITIONS ON POTATO SKIN CATALASE ENZYME
Morgan Winters
ABSTRACT
We tested a particular catalase found in potato skin for changes in function in different
conditions, such as varying pH, catalase concentration, and substrate concentration with and
without an inhibitor. The point of these experiments was to observe the alteration of the
enzyme’s function and to observe the optimal conditions for greatest efficiency. While also
observing changing conditions, we found the Km, the affinity of the substrate and the Vmax, the
maximum reaction rate, which is vital to discovering the optimal conditions and how the enzyme
changes in certain conditions. To find those values, data collected from the lab was used to plot
Michaelis-Mentin and Lineweaver-Burk graphs. Along with the Km and Vmax, these graphs
were used to determine if the inhibitor used, cyanide, is a competitive inhibitor or
non-competitive inhibitor. In the end, it was found that cyanide is a non-competitive inhibitor.
INTRODUCTION
Enzymes are a vital asset to biological systems as they regulate almost all aspects of cellular
reactions. Studying enzymes has proven to be crucial in the biological field, especially medicine,
as it can open many doors, such as how certain diseases work and how to cure them. Our main
study of this experiment was to show the effects an inhibitor has on enzymatic activity. While
this was only focused on potato skin enzymes, it obviously has a wider range of importance. For
example, the enzyme we are focused on is cyanide, a lethal toxin that works to bind to iron in
the enzyme cytochrome C oxidase which prevents the enzyme from transferring electrons to
oxygen in the electron transport chain, effectively cutting off oxygen supply (Graham 2021).
Because of people’s understanding of how cyanide works as an enzyme inhibitor, they were
eventually able to figure out that certain inorganic compounds are able to bind to the cyanide
and become an inhibitor itself. This is just one of many examples of how understanding
enzymatic activity is important for furthering our knowledge, especially in the medical field.
MATERIALS AND METHODS
To start, we prepared the catalase solution using about 5 potatoes, which were then
peeled. Taking the peels, they were placed in a juicer with 4 mL of citrate buffer. The peels were
continuously juiced until we eventually got 25 mL of juice. We then took the juice and filtered it
through a layered cheesecloth soaked in the buffer and drain it into another beaker until we
have at least a total of 20 mls of filtered juice. Next, to prepare the catalase solution, we mixed 1
mL of juice with 4 mL of the 7.2 pH buffer to make a 1:4 dilution. Then, we took 4.5 mL of that
dilution and added .5 mLs more of the buffer in a 50 mL flask
Meanwhile, our partners were setting up the eudiometer by filling a closed burette with
water and inverting it in a 1-liter beaker filled with water. Then after securing the burette with a
clamp, insert the tubing provided into the bottom of the inverted burette. Taking the 50 mL flask
that contains the catalase solution and top with the rubber stopper. We then added 1mL of 30%
hydrogen peroxide into the flask through a 1 mL syringe, slowly. We watched carefully as
bubbles started to form in the tube and started the timer as soon as the first bubble reached the
top of the inverted burette. This procedure was continued up until 2 minutes, where the bubble
reader announced the measurements of the surface of the water every 10 minutes up until a
minute and then every 15 seconds up until the 2 minute mark. While this was preceding, the
swirler was constantly swirling the flask while the recorder was recording the times along with
the measurement readings. As the was just the practice run, this experiment was repeated 4
more times under different conditions
The first variable tested was enzyme concentration. We repeated the creation of the 1:4
catalase buffer dilution but instead using 5 ml of juice to 20ml of buffer instead of just 1:4. To
vary the catalase concentration, the volume of the prepared catalase solution was varied and
mixed with varied amounts of buffer, but always adding up to a total volume of 6 mL. We used
the same eudiometer set up and started repeating the practice procedure with the 25% catalase
concentration going all the way to the 75% concentration.
The next variable tested was different pH conditions. We again prepped the catalase
dilution and then added .9 mL of that dilution with 3.6 mL of varying pH buffers in 5 different
flasks. We again repeated the eudiometer procedure, starting with the 5.8 pH and ending with
the 8.8 pH.
We then tested varying substrate concentrations without an inhibitor. To do this, we
created our normal 1:4 catalase solution but varied the hydrogen peroxide concentration. To
prep the hydrogen peroxide solutions, we took the base 30% hydrogen peroxide and mixed it
with various volumes of water, but all adding up to a total volume of 1 mL. This solution is the
one that is added in by the syringe. We repeated the procedure starting with a 3% concentration
up to the normal 30% concentration
After that, we again varied the substrate concentration but added an inhibitor into the
catalase solution. In this case, .5 mL of cyanide replaces the .5 mL buffer that is usually put into
the catalase solution. We used the same hydrogen peroxide dilutions as in the last experiment.
RESULTS
2- Catalase
concentration
33% (2.0 50% 75%
25% (1.5 mL) mL) (3.0mL) (4.5mL)
Time mL mL mL mL
(sec) Reading O2 Reading O2 Reading O2 Reading O2
0 47.7 0 46.4 0 46.8 0 47.5 0
10 42 5.5 44.9 1.5 42.5 5.1 46.3 1.2
20 38.8 8.7 44.2 2.2 35.7 11.1 43.8 2.5
30 37 10.5 44 2.4 30.5 16.3 41.9 4.4
40 35.5 12 43.7 2.7 27.4 19.4 33 13.3
50 34 13.5 43.5 2.9 24.2 22.6 29.1 17.2
60 32.8 14.7 42.5 3.9 21.7 25.1 25.8 20.5
1:15 31.3 16.2 41.7 4.7 18.7 28.1 21.4 24.9
1:30 29.7 17.8 41.7 4.7 16.2 30.6 17.7 28.6
1:45 28.8 18.9 41.5 4.9 13.9 32.9 14.3 32
2:00 27.9 19.8 40.5 5.9 11.6 35.2 11.4 34.9
Starting with varying enzyme concentration, based on the table we can see that reaction worked
the best at a 75% concentration by the fact that the amount of O2 evolved is at its max for this
experiment. I had expected this to be pretty linear, with more oxygen developing each time the
concentration got higher yet there was a weird instance at 33% where mL of O2 only reached
5.9 mL. However this was do to the fact that something had happened with the tubing and the
gas bubbles were not able to pass through. So under normal circumstances I believe that it
would in fact have a higher O2 volume than 25%.
3- pH
5.8 pH 5.8 pH 6.6 pH 6.6 pH 7.2 pH 7.2 pH 7.8 pH 7.8 pH 8.8 pH
Time
(sec) Reading mL O 2 Reading mL O2 Reading mL O2 Reading mL O2 Reading mL O2
0 48.6 0 47 0 48.3 0 47.5 0 48.5 0
10 46.8 1.8 45.4 1.6 47 1.3 42.5 5 45 3.5
20 46.5 2.1 44.6 2.4 43.5 4.8 41.1 6.4 41.9 6.6
30 46 2.6 43.8 3.2 42 6.3 39.6 7.9 40.4 8.1
40 45.4 3.2 43.3 3.7 41 7.3 39.2 8.3 39.3 9.2
50 45.2 3.4 42.9 4.1 40.4 7.9 38.3 9.2 38.8 9.7
60 44.8 3.8 42.2 4.8 39.9 8.4 38 9.5 38.3 10.2
1:15 44.3 4.3 41.7 5.3 39.1 9.2 37.1 10.4 37.5 11
1:30 43.5 5.1 41.2 5.8 38.5 9.8 36.5 11 37.2 11.3
1:45 43.2 5.4 40.7 6.3 37.8 10.5 36.4 11.1 36.9 11.6
2:00 42.9 5.7 40.4 6.6 37.2 11.1 35.8 11.7 36.4 12.1
With the varying pH, we can see that more mLs of oxygen evolved as the pH got higher, with a
pH of 8.8 with the highest reading of 12.1. The amount of oxygen consisting increased as the
pH had increased. What this means is that the enzyme was the most productive in a basic
environment.
4-
Substrat
e
Concent
ration
4
1 (3%) 2 (7.5%) 3 (15%) (22.5%) 5 (27%) 6 (30%)
Time mL mL mL mL mL mL
(sec) Reading O2 Reading O2 Reading O2 Reading O2 Reading O2 Reading O2
0 47.9 0 48 0 46.4 0 48.9 0 48.8 0 48.4 0
10 44.2 3.7 45 3 40 6.4 40 8.9 40 8.4
20 42.5 5.4 43.5 4.5 36 10.4 35.5 13.4 35.2 13.2
30 41 6.9 41.2 6.8 33 13.4 31.2 17.7 32.8 15.6
40 40.5 7.4 39.5 8.5 31.5 14.9 29 19.9 29.5 18.9
50 39.5 8.4 37 11 28 18.4 26.3 22.6 27.5 20.9
60 38.6 9.3 36.5 11.5 27 19.4 24 24.9 25.5 22.9
1:15 36 11.9 34 14 24.5 21.9 21.8 27.1 23.3 25.1
1:30 35.2 12.7 33.8 14.2 21 25.4 19.1 29.8 21 27.4
1:45 34.2 13.7 32.5 15.5 19.2 27.2 17 31.9 19.6 28.8
2:00 33 14.9 31 17 16.8 29.6 15.5 33.4 18 30.4
Next, we have varying substrate concentration without the inhibitor. First off, I will state that we
again had an incident with the tubing during the 27% substrate concentration, where the gas
bubbles were not able to form or could not pass through due to some physics block, we were
not able to figure out exactly what. Based on the information we have, a concentration of 22.5%
was the most effective for enzymatic activity, as it had produced the most oxygen at 33.4 mL.
Unfortunately this data was not linear, as the 30% concentration had less oxygen than the
22.5%, so I cannot predict whether 27% would have produced more or less oxygen.
5-
Substrat
e
Concentr
ation w/
Inhibitor
4
1 (3%) 2 (7.5%) 3 (15%) (22.5%) 5 (27%) 6 (30%)
Time mL mL mL mL mL mL
(sec) Reading O2 Reading O2 Reading O2 Reading O2 Reading O2 Reading O2
0 49.3 0 49.2 0 49 0 49 0 48.9 0 49.4 0
10 46 3.3 46 3.2 44.5 4.5 41.5 7.5 41.7 7.2 41.5 7.9
20 44.5 4.8 44 5.2 41.7 7.3 37.5 11.5 36.6 12.3 36.5 12.9
30 42.8 6.5 42.5 6.7 39.6 9.4 33.2 15.8 33.8 15.1 33.7 15.7
40 41 8.3 41 8.2 37.5 11.5 31.4 17.6 31.4 17.5 31.5 17.9
50 39.2 10.1 39.5 9.7 35.3 13.7 29.1 19.9 29.1 19.8 29.2 20.2
60 38.5 10.8 38 11.2 32.6 16.4 26.5 22.5 26.9 22 27.2 22.2
1:15 37.6 11.7 36 13.2 29.5 19.5 23.9 25.1 24.5 24.4 25.2 24.2
1:30 37.6 11.7 34 15.2 29.5 19.5 21.6 27.4 22.5 26.4 23.6 25.8
1:45 37.6 11.7 33 16.2 29.5 19.5 20 29 20.4 28.5 21.4 28
2:00 37.6 11.7 32.1 17.1 29.5 19.5 18.3 30.7 19.1 29.8 20.2 29.2
With the varying substrate concentration with the inhibitor, we see that the 27% concentration
was the most effective, albeit barely. However, based on the fact that the 22.5% concentration
was significantly less than the 27% concentration (and even the 30%), it makes me think that
the 27% concentration in the one without the inhibitor would have created more oxygen than the
22.5%.
DISCUSSION
While I did not have an exact prediction on how a more increasingly basic pH would
affect the enzyme function, however I did hypothesize that the data would be linear, which is
proven by our results. These show that the enzyme is the most productive in a basic
environment, yet this is interesting as potatoes tend to be alkaline, even slightly acidic. It makes
me wonder if a potato would grow or thrive the best in a more basic soil. However, just looking
at the table does not give us the whole picture. As I stated previously based on what the table
shows, we would assume that 8.8 pH is the most effective. Yet looking at the Michaelis-Menten
graph, it actually shows that 6.6-7.2 pH is the most effective as it has the highest slope, which is
an indicator for high productivity, even if it didn’t produce the most oxygen in the end. This
shows the importance of having all your data put together and not just to rely on just tables or
just graphs.
For the substrate concentrations, it validated my predictions with the non inhibitor trial
producing considerably more oxygen than the one with the inhibitor. This makes sense as an
inhibitor’s job is to block the enzyme’s productivity. By looking at the graphs made with the data,
we can see that cyanide is non-competitive inhibitor as looking at the Michaelis-Menten graph,
shows that it does in fact affect the Vmax.
LITERATURE CITED
Graham, J. (2021). Cyanide Toxicity. In J. Traylor (Ed.), StatPearls. essay, StatPearls Publishing
LLC.
Suman, S. G. (2019). 14. Chemical and clinical aspects of metal-containing antidotes for
poisoning by cyanide. Essential Metals in Medicine: Therapeutic Use and Toxicity of Metal Ions
in the Clinic, 359–392. https://doi.org/10.1515/9783110527872-020
Pilon, M. et al (2021). BZ 310 Cell Biology Laboratory Manual Fall 2021
FIGURES
Fig. 1.1 Vary pH
Fig. 1.2 Vary Substrate Concentration Without Inhibitor
Fig. 1.3 Varying Substrate Concentration With Inhibitor
Fig. 2.1 Michaelis-Menten of pH
Fig. 2.2 Michaelis-Menten of substrate concentration with and without inhibitor
Without Inhibitor: Km=.460, Vmax= .576
With Inhibitor: Km=.57, Vmax=.521
Fig. 3.1 Substrate concentration without inhibitor
Km=.259, Vmax=.562
Fig 3.2 Substrate Concentration with Inhibitor
Km=.228, Vmax=.492
