Amelia Bailey20.11.09

Mme. ChartrandSBI4UE

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)

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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)

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.

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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 Number: 0 30 60 90 120    

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.

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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.

0 30 60 90 1200

0.5

1

1.5

2

2.5

3

3.5

Trial 1Trial 2Trial 3Trial 4Trial 5

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pH= 1 pH=4 pH=7 pH=10 pH=13Average (Δ mass)(grams): 0.136 -0.488 -0.202 -0.33 -1.124Standard Deviation (grams): 0.404 1.04 0.978 0.358 0.451

0 30 60 90 1200

0.5

1

1.5

2

2.5

3

Series2Series4Series6Series8Series10

0 30 60 90 1200

0.5

1

1.5

2

2.5

3

3.5

4

Series2Series4Series6Series8Series10

6

0 30 60 90 1200

0.5

1

1.5

2

2.5

3

Series2Series4Series6Series8Series10

-30 0 30 60 90 1200

0.5

1

1.5

2

2.5

Series2Series4Series6Series8Series10

7

0 20 40 60 80 100 120 140

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

Change in mass(g) over time (min) when pH=1

Change in mass(g) over time (min) when pH=4

Change in mass(g) over time (min) when pH=7

Change in mass(g) over time (min) when pH=10

Change in mass(g) over time (min) when pH=13

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.136

-0.488

-0.202

0.33

-1.124

Average change in mass (g) over a 120 minute period when pH=1Average change in mass (g) over a 120 minute period when pH=4Average change in mass (g) over a 120 minute period when pH=7Average change in mass (g) over a 120 minute period when pH=10Average change in mass (g) over a 120 minute period when pH=13

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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)

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

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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

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.

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Attachment I: Sample Calculations

Average:

Average=(0.24+0.1+(−0.1 )+0+0.44)/5

Average=0.136 grams

Standard Deviation:

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

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