Amelia Bailey

20.11.09
Mme. Chartrand
SBI4UE
At what pH (1, 4, 7, 10, and 13) does the digestive enzyme pepsin, digest the most protein in
a cube of egg?

Research question:
How will different pH’s (1, 4, 7, 10, and 13) affect the enzyme pepsin’s digestion of proteins in
egg?

Introduction:
pH is a measure of the concentration of hydrogen ions in a solution. The pH scale ranges from 1-
14: a solution with a pH of 7 is neutral, solutions with lower pH value (< 7.0) is acidic, and
solutions with a higher pH value (>0.7) is basic (also called alkaline). The optimum pH of a
solution is the pH at which the working enzyme has maximal activity. The normal gastric pH
(where pepsin is usually found, note: excreted as pepsinogen and mixed with hydrochloric acid
becomes pepsin) is around 2.

Hypothesis:
Pepsin is a digestive enzyme, more specifically; it digests proteins. Pepsin is made up of
pepsinogen (inactive enzyme precursor) and hydrochloric acid, which mixes and activates the
digestive enzyme. It is known that pepsin requires a low pH (as it operates with a high rate of
digestion in the gastric region, which has a very low pH), hence; a highly acidic environment, to
have maximal activity. The indication of the enzymes maximal activity is – in this case – the
decrease in mass, as the protein in the egg (used in this experiment) will be digested, hence; parts
of the egg will dissolve into the solution. For these reasons, the prediction for this experiment is
that the lowest pH’s (1 and 4) will have the largest decrease in mass, while the higher pH’s will
not show a decrease in mass, as the enzyme usually only works in an acidic environment (< 7.0).

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Variables:
Controlled variables:
– Type of eggs used
– The time intervals at which the egg cubes are being massed
– The method of massing the eggs (including drying the pieces, so any residue/solution left
on the egg cube will affect the mass)
– The (approximate) surface area of the egg cubes
– Temperature

Independent variables:
– Different pH’s (1, 4, 7, 10, and 13)

Dependent variables:
– Change in mass of the egg cube

Materials and apparatus:

– Hydrochloric acid (HCl)
– Pepsin
– Distilled water
– Test tubes
– Test tube rack
– Water bath kept at a constant temperature
– Scale (scientific scale)

Procedure (short version):

1. Obtain all materials and apparatus
2. Cut the eggs into approximately equal cubes (surface area and initial mass should be kept
similar)
3. Mix all solutions (5 different samples/test tubes at different pH’s: 5x1, 5x4, 5x7, 5x10,
and 5x13) using the prepared pH solutions and pepsin, and distilled water and HCl for
pH=7. (5 mL of pepsin, 5 mL of HCl, for pH=7: 5 mL distilled water, 5 mL HCl)

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 4. Mass all the egg cubes, before putting one cube into each solution.
5. Mass the egg cubes at intervals of 30 minutes, and make sure you start massing (and
continue massing) in the same order at which you did initially, to ensure correct time
intervals.
6. When massed after 30 min, 60 min, 90 min and 120 min, correctly dispose of all
solutions and remaining content.

Observation Table 1:- Mass (g) of egg cube at different time (min) intervals per pH
- Change in mass (g) after 120 minutes, per trials per pH
- Average difference in mass (g) of all trials per pH
Time: Change in mass (g): Average change in mass (g):
pH: Trial 0 30 60 90 120
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 Number:
1 1 1.88 2.18 2.13 2.16 2.12 0.24 0.136
2 1.77 1.83 1.83 1.88 1.87 0.1
3 2.3 2.24 2.26 2.4 2.2 -0.1
4 2.33 2.39 2.86 2.4 2.33 0
5 1.33 1.81 1.34 1.88 1.77 0.44
4 1 1.56 1.42 1.36 1.91 1.5 -0.06 -0.488
2 2.07 1.73 1.82 1.91 1.88 -0.19
3 2.21 2.02 1.99 1.7 1.55 -0.66
4 2.63 1.85 1.81 1.38 1.35 -1.28
5 1.61 1.46 1.36 1.37 1.36 -0.25
7 1 1.71 1.69 1.7 1.77 1.65 -0.06 -0.202
2 1.45 2.04 2.18 2.19 2.17 0.72
3 2.67 2.81 2.95 3.06 3.08 0.41
4 1.64 1.64 1.73 1.9 1.93 0.29
5 3.42 1.6 1.53 1.71 1.05 -2.37
10 1 1.3 1.43 1.67 1.8 1.82 0.52 0.33
2 1.2 1.31 1.39 1.4 1.38 0.18
3 1.22 1.43 1.51 1.36 1.38 0.16
4 1.6 1.64 1.57 1.95 1.97 0.37
5 2.13 2.34 2.5 2.34 2.55 0.42
13 1 1.33 1.46 1.13 0 0 -1.33 -1.124
2 1.13 1.28 1.37 0 0 -1.13
3 0.92 2 0.73 0 0 -0.92
4 1.42 1.73 1.77 0 0 -1.42
5 0.82 0.9 0 0 0 -0.82
Note! n/a is considered mass=0 grams, as the egg was totally dissolved in the solution, hence the
large decrease in solution pH=13.

Qualitative observations:
The egg cubes in solution pH=1, pH=4, and pH=7 had the same appearance throughout the
whole experiment, while some of the egg cubes in the solution pH=10 and pH=13 turned
somewhat yellow and “mushy” which made it hard to mass. The egg cubes in the two highest
pHs eventually completely dissolved in the solution, leaving no substance left to mass.
Table 3: Average and standard deviation for the change in mass at different pH solutions (1, 4,
7, 10, and 13). See “Attachment 1” for sample calculations.
pH= 1 pH=4 pH=7 pH=10 pH=13
Average (Δ mass)
(grams): 0.136 -0.488 -0.202 -0.33 -1.124
Standard Deviation
(grams): 0.404 1.04 0.978 0.358 0.451

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Conclusion and Evaluation:
This lab did not give the results predicted in the hypothesis, but rather opposite results:
The egg cubes in the solutions pH=13, pH=10 and pH=7 had the largest decrease in mass
(compared to the initial mass, refer to Graph 6: The change in mass (g) over time (min) (120
min) at different pH’s at different trials (±0.05 g)), while the solution where pH=1 only had
small decrease in mass (compared to the initial mass, refer to Graph 6: The change in mass (g)

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over time (min) (120 min) at different pH’s at different trials (±0.05 g)). In the solution pH=1,
which was predicted to be the pH at which the enzyme would be the most active, was shown to
be the solution which decreased the mass by the least.
As solution pH=13 completely dissolved the egg cube, sue to the very high alkalinity of
the solution, the data points for the last trials at that pH had the value n/a, hence; 0. This, of
course, lead to a large decrease in mass when displaying the results graphically. To prevent this
lack of data from occurring, lower pH’s (more acidic solutions) could have been used. This lack
of data points, and the flawed results obtained from the Δ mass = 0 g, caused the trend to be
different than expected, as this was not taken into consideration when planning the lab. If this
was prevented, hence; no extreme decrease in mass due to the egg cube being dissolved in the
solution, the trend would have shown that the pH=13 actually did not work so well, and that
pepsin would not work/work poorly at this pH (theoretically speaking). As the same thing
occurred with some of the pH=10 trials, some of the data obtained from that pH solution is also
flawed, and the same precautions as described for the pH=13 solution must be taken to prevent
these flaws.
Another main error that would very much affect the obtained results, would be the
aqueous residue left on the egg cube when massing it. This extra mass would affect the results
due to the small mass of the cubes, and comparatively large mass of solution left on the cube. To
prevent this, an accurate method of massing and drying the cubes before massing them could be
introduced, and such inaccuracies could be avoided.
A large error would be the time intervals when massing the cubes: the cubes were only
allowed in the solution for 120 minutes, which might not be enough time for the pepsin in some
of the pH solutions to start working. As seen in Graph 1: the change in mass (g) at certain time
intervals (0, 30, 60, 90, 120 min) per trial (1, 2, 3, 4, 5) at pH=1(±0.05 g), the masses are slowly
starting to decrease towards the initial mass, which indicates that if more time was allowed, a
larger increase might have been detected. To prevent this from affecting the results, more time
intervals at a wider range could be used, to ensure accuracy in the results obtained.
A last, but important, error would be the surface area of the egg. Egg cubes with a larger
surface area allows for the enzyme to “attack” it in more places. Hence, the rate of digestion
might increase. Also, a larger surface area leads to greater absorption of the solution, which will
increase the mass by more. To prevent this from happening, a more precise method of cutting the

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cubes could be introduced, or a kitchen appliance (e.g. “Fast Chop”) could be used to ensure
equal cubes, hence; equal surface area.
In conclusion, the results had many errors that heavily affected the results, which
indicates that the results cannot be reliable. This means that even though the results show that
pepsin is most active at pH=4 (mean: 0.488 decrease), pH=7(mean: 0.202 decrease) and pH=1
(mean: 0.136 decrease), this does not have to be the case. In conclusion, the decrease in mass (g)
of solution pH=13 is partially ignored due to the heavy error, and so should most likely pH=10.
This means that these pH’s are not likely to produce maximum enzymatic activity, and hence;
should not be used. The three other pH’s should have some, or none if pH=7, enzymatic activity,
which indicates that these acidic values should be used instead of the alkaline ones. No reliable,
general trend detected, and the experiment was heavily flawed and the results cannot be accurate.

Attachment I: Sample Calculations

Average:
Average=(0.24+0.1+-0.1+0+0.44)/5
Average=0.136 grams

Standard Deviation:

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I) Find the mean (average)
Average=(0.24+0.1+-0.1+0+0.44)/5
Average=0.136 grams

II) Calculate, using subtraction, how much each value differs from the average
Δ=0.136 grams-0.24 grams
Δ=-0.104 grams
...repeat calculation for every value (0.1, -0.1, 0.44, 0)

III) Square the value calculated from II to avoid any negative numbers
Squared Δ=-0.1042
Squared Δ=0.010816 grams
...repeat calculation for every value

IV) Find the mean (average) of the squared values from III
Average=(0.104+0.036+0.236+0.136+0.304)/5
Average=0.1632

V) Take the square root of the value calculated in IV

Standard Deviation=0.1632 grams
Standard Deviation=0.4039801975 grams
Standard Deviation≈0.404 grams