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Table of Contents
EFFECT OF PH ON ENZYME ACTIVITY...............................................................................................1
LOGAN HANSEN, BIOLOGY A1...............................................................................................................1
TABLE OF CONTENTS...............................................................................................................................2
INTRODUCTION..........................................................................................................................................3
METHODS......................................................................................................................................................7
RESULTS........................................................................................................................................................9
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Introduction
The basic function of an enzyme is to increase the rate of a reaction. Most cellular
reactions occur about a million times faster than they would in the absence of an enzyme.
(Ophardt, 2003) Enzymes speed up this rate of reaction so much because they lower the
activation energy that is required for a reaction to occur, therefore allowing it to happen
much quicker than it would on its own. An excellent example of the usefulness of one
without catalase present. When hydrogen peroxide is simply left alone, depending on the
temperature and other factors, it decomposes very slowly. Due to specific properties of
hydrogen peroxide, if it entered the body and was not able to be decomposed quickly, it
would kill cells and cause damage to body systems. However with catalase inside human
body cells, the activation energy needed for the reaction is decreased, and the rate of
decomposition is increased greatly and thus the cells are able to decompose the H2O2 in a
Also the nature of catalase in its ability to break down hydrogen peroxide into
oxygen gas and water gives it several useful medical purposes. If a wound is deep and
has dirt and potentially harmful bacteria in it, we can pour a diluted solution of hydrogen
peroxide in it. Not only is it toxic to the bacteria that are unable to produce catalase, the
catalase in the cells of the human body start to decompose it creating bubbles of oxygen
gas which then push dirt and other contaminants to the surface so as to cleanse and
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One thing that has an effect on the activity of an enzyme is the salt concentration
of the solution that it is in. However this is a little more complex than just having a
change of shape in the enzyme. If the salt concentration is close to zero, the charged
amino acid side chains of the enzyme molecules will attract each other. ..The enzyme will
denature and form an inactive precipitate. If, on the other hand, the salt concentration is
very high, normal interaction of charged groups will be blocked. New interactions will
occur, and again the enzyme will precipitate. ..An intermediate salt concentration such as
that of human blood (0.9%) or cytoplasm is the optimum for many enzymes. (science-
projects.com, n.d.)
Another thing that would have an effect on the activity of an enzyme is substrate
increasing the concentration of the substrate you are increasing the chances of the
substrates coming in to contact with the active site of the enzyme and then being able to
be put into a reaction. However, once that point is reached, the reaction won’t go any
faster because there are only so many enzymes that can catalyze reactions.
reaction will increase drastically. This is because then there are more enzymes to catalyze
the reaction between specific substrates. However, all of the substrates will be used up
(Farabee, 2010) And a change in pH that is too great will denature the enzyme by
changing the shape of the enzyme. (Farabee, 2010) Too much of a change in the shape of
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the enzyme will possibly leave the enzyme functionless, due much to the fact that
enzymes are dependent on their shape for the job that they perform. This scenario is
much the same with temperature, if it is changed too much from the original level, the
enzyme’s shape will change and it will no longer be able to react with the same
substrates.
This then leads to the reason that we are testing the change in activity of catalase
after a change in pH; to determine at what level of change is necessary to give such a
Enzymes are very specific in the reactions that they catalyze. According to our
text book, the active site and the substrates have complementary shapes. The fit is so
precise that the active site and substrates are often compared to a lock and key. (Levine &
Miller, 2003) This theory is often called the Lock and Key Theory. However there is
another theory that is out there that also attempts to explain the reaction between enzyme
and substrate. The induced fit model suggested by Daniel Koshland in 1958 is the more
accepted model for enzyme-substrate complex than the lock-and-key model. (Biology-
online.org, 2008) Unlike the lock-and-key model, the induced fit model shows that
enzymes are rather flexible structures in which the active site continually reshapes by its
interactions with the substrate until the time the substrate is completely bound to it
(which is also the point at which the final form and shape of the enzyme is determined).
(Biology-online.org, 2008)
Generally when the substrates necessary for a reaction come into contact with the
active site of the enzyme the reaction is started, and when this reaction is finished, the
products are released from the enzyme and the whole process can be started again with
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new reactants. This active site is only compatible with specific substrates, this is
explained by the theories above, generally the substrates have to be a perfect or near
perfect fit with the active site, or the enzyme is unable to bond with these materials and
Enzymes are simply proteins, however they are very important to ensure that a
cell functions. When there is a specific reaction that needs to occur in a cell or in an
environment, the nucleus of the cell uses its DNA to code to tell specific parts of the cell
to start producing these proteins that function as enzymes. Like all other proteins,
enzymes are made up of amino acids. They start with a primary structure which is merely
a one-dimensional string of amino acids with a hydrocarbon on one end and a carboxyl
group on the other end. (“Primary Structure,” 2006) This then builds to the secondary
structure which is a three dimensional figure made up of multiple amino acid strings, and
which generally assumes the shape of a helix. (“Secondary Structure,” 2010) And then
into the tertiary structure, upon which the function of a protein (except as food)
depends… and if this is disrupted, the protein is said to be denatured, and it loses its
suggest certain things about the functions and workings of enzymes. First of all, the
enzyme that we are testing is catalase which is found in many different cells throughout
the entire human body. In order to function properly in the human body, the catalase
enzymes would have to be able to function under the conditions that are present in the
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The Purpose of this experiment was to determine the effect that an elevation of
And to determine the pH level at which the catalase works with maximum efficiency.
I believe that the catalase will perform at max efficiency at its original pH level in
the liver. That is I believe that this enzyme will work best without the addition of a base
or acid. In fact, to be even more specific, I believe that the enzyme will work best at a pH
of around 7 due to the fact that that is the pH level that is most commonly found in the
human body which is where the catalase is found, thus reasonably allowing us to
conclude that the catalase would work best at the pH level in which it is found.
Methods
The following procedure was adapted from “The catalyzed rate of decomposition
The first step is to establish the baseline for the hydrogen peroxide. To do this, we
label a syringe H2O2 and draw out 10 mL of hydrogen peroxide from the bottle and put it
into a plastic 60 mL cup labeled baseline. Next using another syringe, we took 1 mL of
distilled water and added it to the hydrogen peroxide solution in the cup. (The distilled
water is to replace the 1 mL of catalase solution that will be used in future trials) Next we
labeled another syringe H2SO4 and drew out 10 mL of sulfuric acid and added that to the
hydrogen peroxide mixture. We then gently swirled the cup so as to completely mix the
contents. Once the solution in the cup was completely mixed, we drew out 5 mL and put
it into another plastic cup labeled titration. We then took that plastic cup and put it under
a burette filled with potassium permanganate suspended on a ring stand. We then opened
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the burette so that the KMnO4 would drip into the contents of our titration cup one drop at
a time. We continued to let the potassium permanganate drip into the cup, swishing the
contents the entire time, until the liquid turned and stayed pink. At which point we closed
the end of the burette so as to stop the flow and then measured the change in the level of
Now that the baseline is established, our next step was to determine how active
the catalase sample is at its normal pH. To do this first we needed to have the catalase
solution. We then took the pH probe and plugged it into the Labquest device and put the
probe into the catalase. We recorded the pH of the liver puree solution as the starting pH.
After taking this measurement we rinsed off the pH probe with distilled water and then
put it in a beaker of tap water to keep it wet. Next we used the H2O2 syringe to put 10 mL
of hydrogen peroxide into another cup. Now with a timer ready, we added 1 mL of the
catalase solution to the hydrogen peroxide and at the same time started the timer. We let
our catalase decompose the hydrogen peroxide for 60 seconds in this experiment. As we
approached the time limit, we drew out 10 mL of H2SO4 and when the time was up, added
the sulfuric acid to the H2O2 and catalase to stop the reaction. After mixing the contents
of the cup, we then proceeded to titrate the mixture of sulfuric acid, hydrogen peroxide
and catalase using the same procedure as above; so as to determine how effective the
Now after both the baseline and neutral pH trials were completed, we proceeded
to put 10 mL of the catalase solution into several different beakers. It is in these beakers
that the sodium hydroxide was added to determine the effect of an elevation of pH on the
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using an eye dropper; and 10 to the next and so on until a pH of 12.5 was reached. After
we added and mixed in the sodium hydroxide into each of the different beakers of
catalase we repeated the steps taken in the trials before substituting in the appropriate
independent variable in this case was the pH of the solution. We chose the number of
drops that were added to the catalase so therefore we controlled this variable thus making
it the independent variable. The dependent variable was the amount of hydrogen peroxide
left after the addition of catalase which translates to the amount of potassium
permanganate used to titrate the solution until it was a pink color. The KMnO4 was used
to measure the hydrogen peroxide content of the solution because when it was added to
the mixture containing the catalase and H2O2, it reacted with the hydrogen peroxide, so
after all of the H2O2 is used up, there is nothing to react with the KMnO4 thus turning the
solution purple. This is dependent because it depends on how active the catalase was
In this experiment, all of the things that we used were reagents. The catalase,
sodium hydroxide, sulfuric acid, hydrogen peroxide and potassium permanganate were
Results
These results model what is happening when the catalase is added to the hydrogen
peroxide and begins to catalyze its decomposition. If we look at table three, the averages,
we see that as the pH increases higher than the start value, the amount of KMnO4 needed
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to titrate the solution increases. This suggests that there is more of the hydrogen peroxide
left over from the reaction and therefore that the catalase was less active in decomposing
the H2O2.
Table Three
Average Change in level of Potassium
Permanganate (Final Level – Initial Level)
Catalase (pH 6.7)Catalase Decomposing Hydrogen
0.8 Peroxide, Trial Two, Table Two
Catalase (pH 8.1) Level of Potassium Permanganate
1.05
Catalase (pH 9.1) Before After
0.9Titration Change in Level of Potassium
Material Titration (mL) (mL) Permanganate
Catalase (pH 11.0) 2.85
Baseline Trial 12.6 16.4 3.8
Catalase (pH 12.5) 3.75
Catalase (pH 6.7) 19.3 20.1 0.8
Catalase (pH 8.1) 20.1 21.2 1.1
Catalase (pH 9.1) 21.2 22 0.8
Catalase (pH 11.0) 22.8 25.6 2.8
Catalase (pH 12.5) 27.6 31.3 3.7
3.5
MnO4 Used Subtracted from Baseline
2.5
1.5
0.5
0
0 2 4 6 8 10 12 14
pH Level of Catalase
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Looking at the graph, there is obviously a negative trend going on after the pH of
the catalase passes a value of around 9.1. However in the range of pH from around 6.7 to
9.1 we see little or no change in the activity of the catalase. This suggests that the catalase
was able to adapt to the change and did not have its shape changed so much as to render
it unable to catalyze the decomposition of the hydrogen peroxide. And when the pH value
reaches around 12.5 we see that the activity of the catalase is just above zero. We know
this because the baseline would be when the y-value of a point is zero, and at a pH of
12.5 the y-value is 0.05 which suggests that barely any of the hydrogen peroxide was
decomposed.
Discussion/Conclusion
When we first started this lab, we wanted to find out what would happen to the
pH of the liquid that it was in. Through the experiment that we devised we explored that
topic and came out with some data that shows trends that can explain what happened to
the enzyme very well. My original hypothesis was that the catalase would perform at
maximum efficiency when it was at or near its original pH level. If we are to look at
figure 1, we see that the catalase is able to keep its regular amount of activity even when
the pH is increased up to around 9.1, we see justification of this when we look at table
three; we see that for these pH values between 6.7 and 9.1 all required around 1 mL of
potassium permanganate for titration. When the pH is elevated to any level higher than
9.1 we see that the activity of the enzyme in decomposing the hydrogen peroxide drops
off very steeply and the activity is just above zero at a pH of 12.5; and when the pH
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increases the amount of potassium permanganate used to turn the solution pink is
drastically increased. And when these amounts of KMnO4 are subtracted from the
baseline we see that they get closer and closer to the established baseline value, which
can be interpreted as the amount of enzyme activity. This drop off can be explained as
being the point at which the shape of the enzyme was changed to such and extreme
amount that it was no longer able to catalyze the decomposition of hydrogen peroxide.
However all of the molecules of catalase’s shapes were not changed when the acid was
added, thus even when the pH was far above its normal level some of the hydrogen
There were many variables in this experiment and almost all of those were. We
controlled the pH of the catalase by adding Sodium Hydroxide to the catalase samples
before they were tested, thus resulting in different pH samples which gave us different
results. Another variable which we controlled was the concentration of the catalase. We
controlled this by diluting the catalase samples that we were given with distilled water to
the concentration that we desired. If the concentration of the catalase was greater than
there would be more enzymes floating about in the solution thus making the chances of
contact between the substrates and the enzymes greater and thereby resulting in a greater
amount of the hydrogen peroxide being decomposed. Yet another variable which was
controlled in this experiment was the amount of time that the catalase was allowed to
react with the hydrogen peroxide. We controlled this by using a stopwatch and sulfuric
acid to stop the reaction. At the same time, the reaction and the stopwatch would be
started and then when the chosen amount of time had passed the sulfuric acid was added
to the sample to stop the reaction from happening. We chose 60 seconds but if the
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allotted time had been longer than more of the hydrogen peroxide would’ve been
decomposed and if the time interval as shortened then of course less of the hydrogen
Before the experiment we thought that the catalase would work best at its original
pH because that was the pH which it had to adapt to working under in the body. Through
the experiment we found that our hypothesis was partially correct. We did indeed find
that the level of activity was near its highest at the original pH; however we also found
that within a range of about 2 on the pH scale the catalase would stay within the range of
its original level of activity. This was not a result which we expected because we thought
that the enzyme would be much more specific in the conditions that it could work under
than we found. So we were not expecting to see such a wide range at which the catalase
stayed at it top level of activity. So as a final concluding note I would say that the data
which we collected in the experiment supports our original hypothesis, but also provides
Despite the fact that we had data that showed a trend that we expected, there were
without doubt errors in our experiment and methods. These errors could throw off the
data and perhaps suggest a trend when graphed that wasn’t truly what happened, or what
should happen. Within the class we saw some examples of this, one of the common
mistakes was that the baseline trial was done incorrectly, and then when the data was
graphed, the line descended into the negatives producing a response that could not be true
for the experiment. Another possible error is with the titration; too much or too little of
the potassium permanganate was used to titrate the solution resulting in inaccurate values
for the change in level of the KMnO4. One of the biggest things that could be done to
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improve the accuracy of this experiment would be simply to do more trials. Also perhaps
adding the potassium permanganate to the solution to titrate it slower would result in
greater accuracy as well because there wouldn’t be to much or too little added to the
solution. Also, to get better data, more catalase could have been tested, because each
sample of catalase that was taken was unique, so if more and more samples were tested,
perhaps the averages for the data would come out to be more accurate.
We especially saw the possibility for error in our experiment on the first day that
we worked on it. We followed much the same procedure on day one with testing the
effect of pH on our catalase activity, however the data that we got was very inaccurate
compared to the data that we collected on day two. If this day one data had been averaged
with the data that was collected on day two then it would’ve acted as an outlier and
skewed all of the data that we were analyzing. For the sake of our data and analysis of
our data, we decided to omit the gathered data from day one in our graphs and tables. I
suspect that many of these flaws in our day one data originated with over titration as
mentioned previously and unfamiliarity with the equipment and the proper way to run the
experiment.
Seeing the data and how it came out left me with some other questions. We saw
from our results that the catalase had a fairly wide range of pH levels that it could
perform at its best at and another question I had was if the same trend would hold for pH
levels below that of normal catalase. Would the activity be high until about a pH of 4
before it dropped to relatively little activity? Also there are different variables that would
be interesting to try such as salt concentration and temperature. Would these provide data
similar to the lab we just did? With the highest activity being in a range around which the
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catalase is normally at, these would all provide interesting areas of study relating to
enzymes. They would also not require very large changes to the existing procedure for
this lab. Simply replace the addition of acid with the addition of salt or the change in
temperature.
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Literature Cited
2008. Effect of salts and inorganic ions on enzyme activity. Retrieved from
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/salt_enz.htm
Miller, Kenneth R. & Levine, Joeseph S. 2003. Biology. Saddle River, New Jersey.
Prentice Hall Publishing. (p. 53)
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