Introduction

Different enzymes function best at different pH levels. Enzymes

in human intestines function best at a pH of around 7.5, while enzymes
in the stomach function best at a pH of about 2 (BBC). When a pH is
either too high or low, the enzyme becomes denatured, making it
unable to catalyze its substrate.
Our goal here is to find out what pH level catalase works best at.
In a previous lab, we tested some pH levels and catalase, but not the
full spectrum. Of the four we tested (3, 5, 7, 9), we found the most
enzyme activity with a pH of 5. Because of this, I believe that there will
be the most enzyme activity, that is, the filter paper will rise up to the
top the fastest, when the pH is around 5.
However, it will also be interesting to note when the enzyme
becomes totally denatured, when there is no enzyme activity and it
does not ever rise to the top of the beaker. Through this experiment we
will be able to be more certain of the actual pH that catalase stops
functioning at. There appeared to be no catalase activity in the
previous lab at pH levels of 3 and 5, so we know that it gets denatured
as the pH becomes more acidic. We do not know when catalase will
become denatured as the solution it is in becomes more basic.

Materials and Methods

Litmus strips
Hydrogen peroxide
Potatoes
Filter paper
pH meter
pH 3,5,7,9 is supplied
Lemon Juice (pH 2)
HCl (pH 0)
7-Up (pH 4)
Root Beer (pH 6)
Mouthwash (pH 8)
Milk of Magnesia (pH 10)
Ammonium Hydroxide (pH 11)
Soapy water (pH 12)
Bleach (pH 13)
Drain cleaner (pH 14)
DI water (pH 7)
Double layered cheesecloth
Funnel

50 mL beakers x 11

500 mL beaker

25 mL test tube

For this lab we first extracted catalase from a potato by cutting it into small
pieces, mixing those small pieces with 150 mL of DI water, and blending it on high for 30
seconds. We then poured this mixture through a double-layered cheesecloth funnel to
collect the filtrate in a beaker. The solution in the beaker was our stock catalase.
We then got 11 different beakers and filled them up with 10 mL of the fluid with
their respective pH levels. We labeled each beaker with its pH. We also filled up a test
tube with 25 mL of H2O2, the substrate of catalase. In this test tube we conducted our
assays.
To conduct our assays, we added 10m mL of our stock catalase to the 10 mL of
whatever solution we were testing, so there was a 50-50 ratio of catalase to fluid. We
soaked a small square of filter paper in the catalase for 5 seconds, tapped it on the edge
of the beaker for 10 seconds to get rid of excess fluid, and then dropped it in the test
tube. We timed how long it took for the piece of filter paper to rise to the top of the tube
again once it was submerged. The faster it raised, the more enzyme activity.
Results
Table 1 Time for Filter Paper to Raise at Varying pH Levels
pH

Trial 1 (sec)

Trial 2 (sec)

Average (sec)

0 (HCl)

Denatured

Denatured

Denatured

2 (Lemon Juice)

Denatured

Denatured

Denatured

4 (Sprite)

80

112

96

6 (Root Beer)

43

45

44

7 (DI Water)

34

39

36.5

8 (Mouthwash)

20

34

27

10 (Mg(OH) )
2

22

23

22.5

11 (NH4OH)

31

27

29

12 (Soapy water)

36

26

31

13 (Bleach)

14 (Drain Cleaner)

Discussion

My hypothesis was not correct, the fastest time actually occurred at a pH of 10.
This contradicts our experience in a previous lab, but we got similar numbers during both
trials at a pH of 10, indicating that a mistake was not made. This likely means that
catalase is most active at slightly higher pH levels, or at least is more effective at basic
pH levels. However, we did not measure the actual pH of our solutions, we just went off
of what pH they were known to have, so it is possible that the actual pH was not 10.
However, due to the purity of our magnesium hydroxide, this is unlikely.
Our results indicate that Catalase is most active at slightly basic pH levels,
showing faster times at pH levels of 8 and 10 (Fig 1, Table 1). They also indicate that
catalase functions much better in basic pH levels than in acidic ones, perhaps most
clearly shown in Figure 1. The speed that the filter paper took to raise to the top
remained somewhat stead once the pH got to 8, but declined steeply from 4 to 8.
When we tested enzyme activity at pH levels of 13 and 14, we got some
surprising results. The filter paper would not even submerge under water; it floated on
top. This could either mean that the enzymes were incredible active, that they were
denatured, or that they became light enough where they would not float. This is definitely
a possibility; solutions with such a high pH level could be damaging to catalase,
removing it from the filter paper, resulting in mostly just paper being put in the solution,
not the catalase. In other words, the high pH levels caused the density of the filter paper
to be less than that of H2O2

References
Fuentes pH Determination of Household Products California State University
Northridge
Pain Free Dentistry "PH Scale." Pain Free Dentistry. N.p., n.d. Web. 29 Nov. 2016.
<http://www.painfreedentistry.uk.com/index.php?
option=com_content&view=article&id=59%3Ataking-the-fear-out-ofdentistry&catid=106%3Adownloads&Itemid=8>.
Forgette, Kyle Quantitative Enzyme Study Dakota County Technical College. Pg. 1, 4.
N.d.