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Enzyme Lab
By: Isabella Haberstock
11/27/15
Period 3

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Introduction
There are many different types of enzymes, and each of them is specific. This means that
each type of enzyme only speeds up one chemical reaction (The Properties of Enzymes). The
enzyme used in this lab is called peroxidase, and it is specific to hydrogen peroxide. When
hydrogen peroxide is poured on the peroxidase, it turns the hydrogen peroxide into oxygen and
water. If you tried to use anything but hydrogen peroxide on peroxidase, absolutely nothing
would happen (What are peroxidase enzymes?). Each enzyme has a little cavity called the
activation site, and only one type of substance will fit there. The enzyme bonds to the substance
and releases it in a less dangerous form (The Properties of Enzymes). In the case of
peroxidase, it bonds to hydrogen peroxide and releases water and oxygen (What are peroxidase
enzymes?). Enzymes are also reusable, which means after a chemical reaction takes place, the
enzyme does not die (The Properties of Enzymes). It will still perform the same function over
and over again as long as it is in its optimal pH and temperature. The optimal pH and
temperature of an enzyme are the pH and temperature where it works best. If the temperature or
pH is too far away from optimal, the enzyme could denature and lose its function. Denaturing is
when the enzyme completely loses its function and cannot do any more chemical reactions
(Enzymes).
In this lab, we will be using Spec 20s to measure the absorbance (rate of chemical
reaction) of each substance. The Spec 20 will shine a light through a test tube, and the substance
absorbs the light and changes color. The substance stops changing colors when the reactant is
gone. In this lab, when all the hydrogen peroxide runs out, peroxidase will stop reacting and the
substance will be stuck at one color. It cannot absorb any more light from the Spec 20s if the

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reaction is not taking place. The darker the final color is, the bigger the absorbance is (Use of
the Spec 20).
In this experiment, we will also be using turnip extract to get the peroxidase. We will also
be using guiacol in this experiment, which is a chemical that reacts with oxygen. When it touches
oxygen, it turns brown. The more oxygen that is produced from the peroxidase reaction, the
darker brown the substance will be. Once the reaction stops, the guiacol will also stop reacting
and the substance will stop changing colors (Enzymes).
The purpose of this lab is to experiment with peroxidase and find out its rate of chemical
reaction and optimal pH. If we test peroxidase with the pHs of 7, 8, 5, 3, and 9, then 8 will be its
optimal pH.

Materials

Test tubes
Guiacol
Turnip extract
Spec 20
Tape and marker for labeling test tubes
Hydrogen peroxide
Tissues
Distilled water
Substances with pHs: 8,9,3,5
timer

Procedure
1. Plug in Spec 20s and make sure the wavelength is at 460 and absorbance is 0
2. Invert control test tube (8.9 mL distilled water, .1 mL guiacol, 1 mL turnip extract) and
put in slot of Spec 20
3. Keep absorbance at 0
4. Make test tubes 2 and 3
5. Test tube 2 (substrate)- .1 mL guiacol, .2 mL hydrogen peroxide, 4.7 mL distilled water
6. Test tube 3 (enzyme)- 1 mL turnip extract, 4 mL distilled water

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7. Get phone timer ready
8. Mix test tubes 2 and 3, start timer, and invert contents
9. Wipe tube with tissue, take control tube out of Spec 20 and put new tube in
10. Record absorbance every 20 seconds for the first minute and every thirty seconds for the
remaining nine minutes under run 1
11. Put control test tube back in Spec 20
12. Reset timer
13. mix together test tube 2, test tube 3, and test tube substance with pH
14. invert tube, wipe off, and put in Spec 20
15. repeat steps 10 to 14 for each pH

Results

Absorbance of Peroxidase
2.5
2
pH 7

1.5

Absorbance

pH 8
pH 5

pH 3
pH 9

0.5
0

Time

This graph shows the rate of chemical reaction of peroxidase for the pHs of 7, 8, 5, 3, and
9. The y-axis shows the absorbance given by the Spec 20 at certain intervals, and the x-axis
shows all the different time intervals.
Discussion
The pHs of 7, 5, 8, and 9 seemed to have worked on peroxidase, but pH 3 is not the
optimal pH because it barely reacted at all and only rose a little above the x-axis. My

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interpretation of this graph is that pH 7 is the optimal pH for peroxidase. The other pHs of 5, 8,
and 9 did react, but pH 7 reacted quickly and hit the maximum absorbance that the Spec 20 can
calculate before the other pHs. Even though pH 8 still reacted almost as well as pH 7, my
hypothesis was incorrect.
The major source of error in this experiment was timing. We wrote down the absorbance
a few seconds after each interval because it was constantly changing, and we almost missed a
few intervals. We also did not wipe the test tube very well before putting it in the Spec 20
because we only had a few seconds to do so, which could have interfered with how much light
shined through the test tube.
If we do more experiments on peroxidase in the future, then we could test other pHs that
are more basic. There was a very acidic pH of 3, but the most basic substance we tested had a pH
of 9. We could also repeat this experiment, but instead of using different pHs, we could use
different temperatures to find the optimal temperature of peroxidase.
References
Enzymes. Nku.edu. NKU. Web. 27 November 2015.
The Properties of Enzymes. Abpi. The Association of the British Pharmaceutical Industry,
2015.
Web. 27 November 2015.
Use of the Spec 20. Bates.edu. Bates College, 2002. Web. 27 November 2015.
What are peroxidase enzymes? wiseGeek. Conjecture Corporation, 2015. Web. 27 November
2015.