Investigating the Effect of Enzyme
(Peroxidase) Concentration on Reaction Rate
Introduction
A chemical reaction is when two or more molecules (the reactants) interact with each other and are
rearranged to form different molecules (the products). Chemical reactions happen constantly
throughout the universe. There are many different types of chemical reactions including: reduction
and oxidation, precipitation, combination and decomposition. Chemical reactions can be sped
up/slowed down in a number of different ways including: temperature, agitation, increased
concentration, surface area and catalysts. Chemical reactions can be sped up by catalysts; however,
the catalyst is not used in the chemical reaction. Enzymes are biological catalysts which are used
frequently in living things.
In this experiment the enzyme peroxidase, derived from turnips, was used to speed up the chemical
reaction between Hydrogen Peroxide and Guaiacol to produce Tetraguaiacol and water. Guaiacol
and Hydrogen Peroxide is clear, however, Tetraguaiacol is orange; therefore, the rate of the reaction
was measured on how fast the solution turns orange. This colour-changing rate will be measured by
a colorimeter. This can be illustrated in the chemical equation:
4H2O2
+
Hydrogen Peroxide
(Clear)
4C7H8O2
Guaiacol
(Clear)
--------------->
Enzyme
Peroxidase
(C7H8O2)4
Tetraguaiacol
(Orange)
+
H2O
Water
(Clear)
A colorimeter, in this case, measured absorbance. The colorimeter works by shining a beam of light
through the solution and then records the about of light on the other side- this measures how much
light has been absorbed. As the solution turns orange the solution’s absorbance will increase and will
be measured by the colorimeter (as seen in figure 1).
Figure 1: A Simplified Diagram Of How A Colorimeter Works
Aim
To investigate the effect of an enzyme on the rate of reaction of Hydrogen Peroxide and Guaiacol.
Hypothesis
The higher the concentration of peroxidase the faster the reaction rate.
Levi Burgess | 10.4
Variables
Independent
Enzyme (Peroxidase) Concentration (mL)
Dependent
Absorbance
Constants




Amount of pH 5 Buffer- 1mL in each reaction
Amount of Hydrogen Peroxide (H2O2)- 0.5mL in each reaction
Amount of Guaiacol (C7H8O2)- 0.25mL in each reaction
Temperature (20oC)
Materials












A sample of pH 5 Buffer
A sample of Dilute Hydrogen Peroxide
A sample of Dilute Guaiacol
A sample of pH 7 Phosphate Extraction Buffer
A sample of Peroxidase (enzyme)
Colorimeter
18 Cuvettes (3 for each concentration)
Stopwatch
Electronic Pipette (Which can measure to 0.000 of a millilitre)
5 Pipette nozzles (one for each chemical)
Normal Pipette
Tissues
Method
1. Turn on colorimeter, adjust wavelength setting to 500 nm, and allow instrument to warm up
for 15-20 minutes.
2. Prepare two cuvettes for each Reaction: one containing reactants (Cuvette R) and the other
containing the enzyme (Cuvette E)
a. Table 1: Reaction 1:
Cuvette R
Cuvette E
0.5 mL pH 5 buffer
0.5 mL pH 5 buffer
0.5 mL Dilute Hydrogen Peroxide
1.125 mL pH 7 Phosphate Extraction Buffer
0.25 mL Dilute Guaiacol
0.125 mL Peroxidase
b. Table 2: Reaction 2:
Cuvette R
Cuvette E
0.5 mL pH 5 buffer
0.5 mL pH 5 buffer
0.5 mL Dilute Hydrogen Peroxide
1.025 mL pH 7 Phosphate Extraction Buffer
0.25 mL Dilute Guaiacol
0.225 mL Peroxidase
c. Table 3: Reaction 3:
Cuvette R
0.5 mL pH 5 buffer
0.5 mL Dilute Hydrogen Peroxide
0.25 mL Dilute Guaiacol
Cuvette E
0.5 mL pH 5 buffer
0.925 mL pH 7 Phosphate Extraction Buffer
0.325 mL Peroxidase
3. Prepare the control cuvette by combining: 1mL pH 5 buffer, 0.5 mL Dilute Hydrogen
Peroxide, 0.25mL Dilute Guaiacol and 1.25mL pH 7 Phosphate Extraction Buffer.
Levi Burgess | 10.4
4. Zero the colorimeter (zero absorbance) at 500 nm using the control solution
5. When ready to begin, carefully pour contents of reaction 1’s cuvette R into cuvette E, stir
with pipette and wipe clear side of cuvette with a tissue. Then place it into the colorimeter
and immediately start timing.
6. Measure and record the absorbance as a function of time every 30 seconds for 5 minutes.
7. Repeat step 5-6 for each reaction.
8. Plot the results on a graph (Absorbance v Time, reaction rate is slope of the line)
Figure 2: Experimental Setup
Figure 3: Combining The Cuvettes
Safety
A few of the chemicals used are minor irritants. Dilute Guaiacol is slightly toxic and is harmful if
inhaled and could cause skin irritation if it comes in contact with exposed skin; therefore, it would be
prudent to replace the lid on the Guaiacol when it is not being used and to wear a lab coat, glasses
and gloves to protect eyes and skin. Hydrogen peroxide is an oxidiser and bleach so it can also cause
Levi Burgess | 10.4
skin irritation and is harmful if swallowed or breathed in, so follow above precautions. There is also
glassware used in this experiment so be careful not to break it and keep it away from the edge of
the work bench.
Results
Table 4: Light Absorbance in relation to amount of Peroxidase over 5 minutes
Time (sec)
Concentration 1
Concentration 2
Concentration 3
(0.125mL)
(0.225mL)
(0.325mL)
0.261
0.279
0.323
0.355
0.392
0.428
0.461
0.493
0.524
0.552
0.581
0.054
0.098
0.153
0.208
0.245
0.305
0.347
0.393
0.425
0.461
0.496
0.800
0.855
0.920
0.950
1.080
1.090
1.189
1.210
1.267
1.287
1.288
0
30
60
90
120
150
180
210
240
270
300
Graph 1: Reaction Speed of Hydrogen Peroxide
and Guaiacol with varying amounts of Peroxidase
1.6
1.4
y = 0.0018x + 0.8197
Absorbance
1.2
1
0.8
y = 0.0011x + 0.2572
0.6
0.4
y = 0.0015x + 0.0645
0.2
0
0
50
100
150
200
250
300
350
Time (Seconds)
Concentration 1 (0.125mL)
Concentration 2 (0.225mL)
Concentration 3 (0.325mL)
Blank
Discussion
The results show that the amount of peroxidase does change the reaction rate. The hypothesis that
‘the higher the concentration of peroxidase the faster the reaction rate’ is supported by the results
(see Graph 1). It is evident (as seen in graph 1) that the higher the concentration of Peroxidase, the
higher the absorbance became in a far shorter period of time; therefore, the reaction occurred
Levi Burgess | 10.4
quicker- supporting the hypothesis. This is especially evident in Reaction 3’s steep linear trend line
compared to the other concentrations; as Reaction 3 had the most enzyme in it (0.325mL compared
to 0.225mL and 0.125mL), it reacted the quickest. The control, which had no enzyme in it, did not
react at all (see graph 1), showing that the presence of an enzyme is required for the reaction to
occur at any significant speed.
If a solution contains a higher concentration of enzymes, then the reaction rate will increase because
there are more enzymes to break down the substrate (substance on which an enzyme acts).
Therefore, the more enzymes present the quicker the substrate will be broken down and in turn, the
quicker the reaction- as supported by the results. More enzymes also means that more collisions
with reactants will occur per unit of time, hence, a faster conversion of reactants to products.
However, one would assume if this experiment continued on long enough the reaction rate will
eventually plateau as the enzyme molecules become saturated (when all the molecules of enzymes
are working at full capacity); however, the more enzyme molecules the longer it will take for them to
become saturated, therefore, the quicker the reaction. The reaction rate will also plateau when all
the Hydrogen Peroxide and Guaiacol molecules become Tetraguaiacol and water molecules, as
illustrated in the chemical equation: 4C7H8O2 + 4H2O2 C28H24O8 + H2O The effectiveness of enzymes
are affected by temperature and pH, which is why a pH buffer was used to keep the solution at the
optimum pH, and the temperature was room temperature; this isolated the effect of the enzyme
purely to its concentration- the independent variable (Chemistry for Biologists, 2014).
A random error is one made by human limitations and potentially affects each test differently. One
potential random that was not mixing the solution properly before putting it into the colorimeter. If
the solution was not mixed enough it: would affect the absorbance as the colorimeter is very precise
and would take longer for the reaction to take place, since the enzyme would not have been
thoroughly mixed with the other reactants. More trials could be conducted to reduce the effects of
this random error.
Another potential random was the fact that the time varied between mixing Tube R and Tube E
before placing it in the colorimeter in each trial. The reaction began as soon as the cuvettes were
mixed so waiting different periods of time between measuring the absorbance would have given
different readings. This may explain the excessively high trend line for Concentration 3 (see graph 1)
since this reaction would have happened quicker than the other two, as there was more enzyme, so
the wait would have had a larger effect. This may also explain why Concentration 1’s trend line is
higher than Concentration 2’s (see graph 1), since the wait may have been longer for concentration
1 so the results are higher. However, the rate of reaction is based measured of the gradient of the
line and Concentration 2’s line is steeper so the rate of reaction was quicker- supporting the
hypothesis. One way to reduce the effect of this random error would be to have a set period of time
between mixing the solutions and taking the first reading. More trials would minimise the effects of
these random errors.
A systematic error is an error caused by imperfections in the equipment used, systematic errors
unlike random errors affect all the trials equally. One potential systematic error was the colorimeter
not being calibrated properly. This would affect all the results by a certain amount. When the results
are graphed the systematic error would affect the vertical translation of the results graphed,
however, not the slope of the lines, therefore not the rate of the reaction. This systematic error
could be identified by repeating the experiment with different equipment and then comparing the
results.
Only one trial of each concentration was recorded, therefore, any random errors would have had a
large impact on the results, and would have been difficult to identify. Therefore, one major
improvement to the method would be to do each concentration at least three times so that the
results can be averaged, in turn reducing the effect of the random errors. Another improvement
Levi Burgess | 10.4
would be to have a set time limit between mixing the solutions and taking the first reading, this
would help keep the results consistent and, therefore, precise.
Conclusion
The aim of this experiment was to investigate the how the concentration of the enzyme (peroxidase)
affects the reaction rate. The hypothesis was that the higher the concentration of peroxidase the
faster the reaction rate. By using a colorimeter, stop watch, Hydrogen Peroxide, Guaiacol, pH 5
Buffer, pH 7 Phosphate Extraction Buffer and varying concentrations of the Peroxidase enzyme, the
results showed that the enzyme concentration does affect the reaction rate, and that the stated
hypothesis was supported. The reaction rate increased and decreased with the concentration of
Peroxidase; the higher the concentration the more enzymes present in the solution, the more
enzymes present the faster the reactants are rearranged, the faster the reactants are rearranged the
quicker the reaction rate. The experiment and its results would be more reliable if further trials were
undertaken.
Bibliography

Levi Burgess | 10.4
Chemistry for Biologists, (2014). Enzymes. [online] Available at:
http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm [Accessed 10 Sep.
2014].