Peroxidase Enzyme Activity

Joseph Verry
AP Biology
12/1/16
Period 4
Abstract:
 For my experiment enzyme test, I decided to test whether adding a heavy metal like
copper to a hydrogen peroxide decomposition reaction would increase or decrease the rate of
reaction. I followed the baseline activity first for control data and then added 0.5 M Copper (II)
Sulfate to one of the reactions. I hypothesized that the reaction would decrease and the data
coincided with my prediction.
Introduction:
Enzymes are the catalysts of biological systems. Enzymes and catalysts increase the rates
of biological or chemical reactions by decreasing the activation energy required for a reaction
and providing a lower energy pathway from reactants to products. Peroxidase enzymes are
widely distributed in animals, plants, and bacteria, to protect cells against the effects of oxidative
stress and cell damage due to hydrogen peroxide (Source 2). The peroxidase was extracted from
turnips and provides a model enzyme for studying enzyme activity. The rate of an enzyme-
catalyzed reaction depends on biotic and abiotic factors, such as enzyme and substrate
concentration, pH, temperature, and the presence of inhibitors and activators. Will the Cu2+ ions
that dissociate from Copper(II) Sulfate inhibit the ability of peroxidase to react with hydrogen
peroxide? If the Copper (II) Sulfate is added into the Peroxidase Enzyme reaction, then the rate
of decomposition will be reduced because copper is a common heavy metal noncompetitive
inhibitor of many enzymes.
Materials:
1. Buffer, pH 5
2. Guaiacol, 0.2% solution
3. Hydrogen Peroxide, 0.02% solution
4. Phosphate Buffer, pH 7
5. Peroxidase Enzyme Extract
6. Pipet Bulb and Pipets, serological, 2 mL
7. Spectrophotometer
8. Test tubes
9. Test Tube rack
10. Timer
11. Cu2SO4, 0.5% solution

Procedure:
1. Complete the Baseline Activity in order to have data to compare to following experiment.
2. Turn on the spectrophotometer, adjust the wavelength setting to 470 nm, and allow the
instrument to warm up for 15-20 minutes.
3. Prepare a “blank” by combining 4 mL pH 5 buffer, 2 mL 0.02% hydrogen peroxide, 1
mL 0.2% guaiacol, and 2 mL phosphate buffer in a test tube.
4. Put the “blank” in a covette and use it to calibrate the spectrophotometer at 470 nm.
5. Prepare one test tube with 1 mL of the pH 5 buffer, 2 mL of the 0.02% H202, 1 mL of
0.2% Guaiacol, and 0.5 mL of the 0.5% Cu2SO4.
6. Prepare one test tube with 2 mL of the pH 5 buffer, 1 mL of the Enzyme Extract, and 1
mL of the Phosphate Buffer.
7. Mix the two test tubes prepared in steps 4 and 5 and pipet a small amount into a covette.
Put the filled covette into the spectrophotometer and start recording the first absorbance
readings.
Data:
➢ Part 1(Baseline Activity):
 Trial 1 Trial 2 Trial 3

Slope of Line 0.001495 0.001823 0.0003188

➢ Part 2(Personal Experiment):

Trial 1

Slope 0.0005040

Results and Discussion:

Since my experiment was a reproduction of Trial 2 in the Baseline Activity with Copper
(II) Sulfate added to the reaction I will compare the slope of Trial 2 with the one trial done for
my experiment. In Trial 2 of the baseline activity, the slope is 0.001823 while Trial 1 in my
personal experiment is 0.0005040. This coincides with my hypothesis because the rate of the
reaction involving copper ions is about half of the reaction without. These conclusions lead me to
accept my hypothesis because I predicted that the introduction a heavy metal would inhibit the
decomposition reaction of hydrogen peroxide. This applicable to real life because it shows one
way a high concentration of a heavy metal like copper can inhibit an important reaction from
occurring in plants. Some sources of error would be that Copper (II) Sulfate is colored blue and
also is not water soluble so this factor before the reaction begins started the reaction around 0.8
absorbance.
Conclusion Questions:
A. Do metal ions activate or inhibit the rate of enzyme-catalyzed decomposition of hydrogen
peroxide?
B. If the Copper (II) Sulfate is added into the Peroxidase Enzyme reaction, then the rate of
decomposition will be reduced because copper is a common heavy metal noncompetitive
inhibitor of many enzymes. (Source 1)
C. To test this hypothesis, I will repeat Trial 2 from the Baseline activity but also add 0.5
mL of 0.5M Copper (II) Sulfate to the reaction and record the data and compare to the
baseline activity.
D. Same safety procedure as the Baseline Activity which includes goggles and standard lab
procedure of washing hands before and after lab. Also, the concentration of Copper is so
miniscule that it should be safe to just pour the contents of the lab from the test tubes
down the drain with plenty of water.
E. I will record the data using a spectrophotometer calibrated at 470 nm, just as I did in the
baseline.
F. I will compare the slope of the line created by graphing absorbance over time for my
experimental trial with our slope for the baseline activity.
G. N/A
H. The data supports my hypothesis because the rate of the decomposition reaction with
Copper (II) Sulfate was lower than the reaction without.
I. According to the data, heavy metals are noncompetitive inhibitors and in this case,
Copper prevented Peroxidase from functioning properly. This is caused by copper
bonding to the protein, not at the active site, causing there to be a change in structure and
then causing the enzyme not to function properly (Source 3).
J. Test other heavy metals like lead, mercury, and silver and see if the results are the same
as the data collected on Copper. It could also be possible to test catalase and see if this
enzyme is affected by heavy metals as well, showing how heavy metals can poison
humans as well.
Bibliography:
1. Clark, Jim. "Enzyme Inhibitors." Enzyme Inhibitors. N.p., May 2016. Web. 06 Dec. 2016.
2. "Enzymes." Enzymes. N.p., n.d. Web. 06 Dec. 2016.
<http://www.nku.edu/~whitsonma/Bio150LSite/Lab%2011%20Enzymes/Bio150LEnzymes.html>.

3. Enzymes. ENZYME REACTIONS–Peroxidase I – BACKGROUND Introduction: (n.d.): n. pag.

Web. <http://www.indiana.edu/~l113/independent_projects/addlprocedures/AP-VEnzymes.pdf>