Universiti Tunku Abdul Rahman (Kampar Campus)

Faculty of Science, Engineering, and Technology

Bachelor of Science (Hons) Biotechnology

Year 2 Semester 1

UESB 2142 Laboratory 2A

(II) Biochemistry and Cell Metabolism

Lecturer: Mr. James Wee Sheau Yang

Student’s Name: Cheah Hong Leong

Student’s ID: 08AIB03788

Experiment No. 3 and 4

Title: Isolation of the Enzyme Peroxidase from Radish and Properties of

Peroxidase.

Date: July 13, 2009

Title: Isolation of Peroxidase from Radish and Properties of Peroxidase

Objectives:
– To extract the enzyme Peroxidase from the radish.
– To determine the effect of concentration of enzyme Peroxidase on the rate of
reaction based on the optical density obtained through spectrophotometer.
 – To compare the rate of reaction catalyzed by crude Peroxidase with that of the
commercial Peroxidase.

Results:
Table 1: Optical Density Readings over Time Obtained from Spectrophotometer on Five
Test Tubes

Time, Optical Density, A

s Test tube Test tube Test tube Test tube Test tube
1 2 3 4 5

5 0.265 0.273 0.145 0.119 0.100

10 0.275 0.340 0.176 0.120 0.126
15 0.285 0.414 0.208 0.121 0.147
20 0.312 0.470 0.242 0.122 0.170
25 0.325 0.503 0.279 0.123 0.191
30 0.340 0.520 0.311 0.125 0.214
35 0.358 0.535 0.347 0.126 0.231
40 0.372 0.546 0.380 0.127 0.250
45 0.387 0.558 0.412 0.128 0.274
50 0.398 0.572 0.442 0.128 0.286
55 0.410 0.584 0.478 0.129 0.300
60 0.421 0.597 0.502 0.130 0.318
65 0.436 0.610 0.526 0.131 0.333
70 0.449 0.624 0.554 0.132 0.349
75 0.461 0.636 0.582 0.133 0.360
80 0.478 0.648 0.604 0.133 0.374
85 0.490 0.661 0.630 0.134 0.385
90 0.501 0.674 0.650 0.134 0.395
95 0.508 0.685 0.670 0.135 0.408
100 0.519 0.698 0.687 0.136 0.419
105 0.528 0.709 0.703 0.136 0.429
110 0.537 0.721 0.719 0.137 0.440
115 0.549 0.732 0.734 0.137 0.450
120 0.560 0.742 0.748 0.138 0.460
125 0.568 0.754 0.760 0.138 0.466
130 0.576 0.763 0.770 0.139 0.474
135 0.584 0.775 0.780 0.139 0.481
140 0.593 0.783 0.789 0.140 0.489
145 0.601 0.792 0.800 0.140 0.498
150 0.608 0.800 0.806 0.140 0.505
 155 0.615 0.808 0.814 0.141 0.515
160 0.623 0.814 0.820 0.141 0.522
165 0.630 0.822 0.827 0.141 0.528
170 0.636 0.828 0.833 0.141 0.533
175 0.642 0.834 0.839 0.142 0.537
180 0.648 0.839 0.843 0.142 0.542
*The contents in the test tube from 1 to 5 were refers to Laboratory Manual UESB 2142
Laboratory 2A (I) Biochemistry and Cell Metabolism page 7.
*Graph 1 to 5 in attachments were plotted based on the table above and the slope of the
graphs were calculated to obtain the zero order reaction rates.
*Optical density does not have true unit; Absorbance, A is used.

Zero order reaction rate calculation:

Graph 1: Optical Density versus Time for Test Tube 1
Gradient of the graph = 0.23A/84s
= 2.74 x 10-3As-1
Zero order reaction rate = 2.74 x 10-3 As-1
Graph 2: Optical Density versus Time for Test Tube 2
Gradient of the graph = 0.2A/78s
= 2.56 x 10-3As-1
Zero order reaction rate = 2.56 x 10-3 As-1
Graph 3: Optical Density versus Time for Test Tube 3
Gradient of the graph = 0.31A/46s
= 6.74 x 10-3As-1
Zero order reaction rate = 6.74 x 10-3 As-1
Graph 4: Optical Density versus Time for Test Tube 4
Gradient of graph = 0.01A/55s
= 1.82 x 10-4As-1
Zero order reaction rate = 1.82 x 10-4 As-1
Graph 5: Optical Density versus Time for Test Tube 5
Gradient of graph = 0.34A/76s
 = 4.47 x 10-3 As-1
Zero order reaction rate = 4.47 x 10-3 As-1
Calculation on concentration of Peroxidase:
Weight of dried crude Peroxidase = 0.0204g
= 20.4mg
Volume of distilled water added = 10ml
Concentration of crude Peroxidase = 20.4mg/10ml
= 2.04mg/ml
Test tube 1- M1V1 = M2V2
(2.04mg/ml)(2.0ml) = M2 (3.2ml)
M2 = 1.275mg/ml
Test tube 2- M1V1 = M2V2
(2.04mg/ml)(1.0ml) = M2 (3.2ml)
M2 = 0.638mg/ml
Test tube 3- M1V1 = M2V2
(2.04mg/ml)(0.5ml) = M2 (3.2ml)
M2 = 0.319mg/ml
Concentration of commercial Peroxidase = 10mg/100ml
= 0.1mg/ml
Test tube 5- M1V1 = M2V2
(0.1mg/ml)(1.0ml) = M2 (3.2ml)
M2 = 0.031mg/ml

Table 2: The Zero Order Reaction Rate and the Concentration of Enzyme Peroxidase

Test Peroxidase concentration, Zero Order Reaction Rate, x

Tube mg/ml 10-3 As-1
1 1.275 2.74
2 0.638 2.56
3 0.319 6.74
 4 0.000 0.18
*5 0.031 4.47
*The Peroxidase used in test tube 5 was commercial Peroxidase.
*Graph 6 in attachment was plotted based on the above table.

Conclusion and Discussion:

By theory, the zero order reaction rates between hydrogen peroxide and p-
phenylenediamine should be directly related to the concentration of peroxide that
catalyzes the reaction.
However from Graph 3, the zero order reaction rate obtained from Test tube 3 was
extremely highest among the Test Tube 1, 2, and 3 even though the concentration of the
peroxide in Test Tube 3 was the lowest. This indicates that some error(s) had occurred
when the experiment was in progress.
However, since all the crude preparation of diluted Peroxidase solution in the three test
tubes were taken from the same source of crude Peroxidase solution, possibility of error
in Experiment 3 was eliminated.
The only possible errors might happen only in Experiment 4. One of the possible errors
might be the contamination of cuvette by Peroxidase from Test tube 1 and 2. The
structure of cuvette tends to trap some solution inside it even after rinsed with distilled
water. Additional of Peroxidase in little amount in cuvette can cause high increase in
reaction rate as enzyme is only needed in little quantity.

When comparing the crude peroxide in Test Tube 1 and 2, the Peroxidase concentration
in Test tube 1 was two-fold than that of in Test Tube 2. However, the zero order reaction
rate calculated in Test Tube 1 was only slightly higher than that of Test Tube 2.
This might cause by the limitation effect of the concentration of either hydrogen peroxide
or p-phenylenediamine or both of them. Peroxidase might be needed in little amount to
catalyze the reaction, over excess of quantity of Peroxidase did not further increased the
reaction rate.
From Graph 4, little increase in optical density obtained from the spectrophotometer
indicated that there was still has reaction of hydrogen peroxide and p-phenylenediamine,
even though lack with Peroxidase.
One possible explanation was the contamination or presence of little amount of inorganic
catalyst in water or any other solutions being added together into the test tube. The
inorganic catalyst presence in little amount can be metallic ions such as copper ion, Cu2+,
manganese dioxide, and silver ion.
From the Graph 5 and Graph 6, the zero order reaction rate catalyzed by commercial
Peroxidase was much higher than that of crude preparation of Peroxidase. From Graph 6,
the concentration of commercial Peroxidase was only 0.031mg/ml, which was much
lower than crude Peroxidase concentration in Test Tube 1, 2, and 3. However, the zero
order reaction rate in Test Tube 5 was much higher than that in Test Tube 1 and 2.
One possible explanation for this result was the impurities of crude preparation of
Peroxidase in Experiment 3. Although the concentration of crude enzyme in Test Tube 1
and 2 seemed to be higher than concentration of commercial enzyme in Test Tube 5,
however, any possible improper techniques used in extraction of Peroxidase from radish
might cause impurities in the extracted enzyme solution. The extracted enzyme might
contain largely of other compounds presence in the radish cells and only little amount of
Peroxidase.
Another possible reason was the preparation techniques in commercial industry were
much sophisticated.
References:
Campbell, N.A. & Reece, J.B., 2005. Biology. 7th ed. CA: Pearson Benjamin Cummings.
Voet, D.J., Voet, J.G., & Pratt, C.W., 2008. Principles of Biochemistry. 3rd ed. NJ: John
Wiley & Sons, Inc.
Wikipedia 2009, Hydrogen Peroxide, online, retrieved 25 July 2009, from
http://en.wikipedia.org/wiki/Hydrogen_peroxide
Wikipedia 2009, Peroxidase, online, retrieved 25 July 2009, from
http://en.wikipedia.org/wiki/Peroxidase
Attachments:
Graph 1: Optical Density versus Time for Test Tube 1
Graph 2: Optical Density versus Time for Test Tube 2
Graph 3: Optical Density versus Time for Test Tube 3
Graph 4: Optical Density versus Time for Test Tube 4
Graph 5: Optical Density versus Time for Test Tube 5
Graph 6: Zero Order Reaction Rate versus Peroxidase Concentration