NAME : LOVINIA A/P SUGUNASILAN

ID NUMBER : 09ANB07210

LABORATORY 2A : BIOCHEMISTRY AND CELL METABOLISM

COURSE : BIOTECHNOLOGY (YEAR 2 SEM 3)

TUTOR’S NAME: MR. JAMES WEE

DATE: 17TH MARCH 2011

EXPERIMENT 3: THE ISOLATION AND PROPERTIES OF ENZYME

PEROXIDASE FROM RADISH

TITLE:

The isolation and properties of enzyme peroxidase from radish.

OBJECTIVES:

- To learn how to prepare a crude preparation of peroxidase

- 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 between crude preparation and commercial peroxidase
INTRODUCTION:

The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively

in biochemistry applications primarily for its ability to amplify a weak signal and increase
detectability of a target molecule. Horseradish peroxidase is a 44,173.9-dalton glycoprotein with
4 lysine residues for conjugation to a labeled molecule. It produces a coloured, fluorimetric, or
luminescent derivative of the labeled molecule, allowing it to be detected and quantified. HRP is
often used in conjugates (molecules that have been joined genetically or chemically) to
determine the presence of a molecular target. For example, an antibody conjugated to HRP may
be used to detect a small amount of a specific protein in a western blot. Here, the antibody
provides the specificity to locate the protein of interest, and the HRP enzyme, in the presence of
a substrate, produces a detectable signal.[1] Horseradish peroxidase is also commonly used in
techniques such as ELISA and Immunohistochemistry.

Horseradish peroxidase is ideal in many respects for these applications because it is

smaller, more stable, and less expensive than other popular alternatives such as alkaline
phosphatase. It also has a high turnover rate that allows generation of strong signals in a
relatively short time span.

In plant systems, peroxidase is likely to play a role in synthesis of the plant cell wall.
Here, the enzyme cross-links phenolic residues of cell wall polysaccharides and glycoproteins,
which serve to strengthen the cell wall components. This action may represent part of a wound-
healing response because some peroxidase isoenzymes are induced by stress such as that caused
by high salt, physical wounding and microorganisms.

In this case, the peroxidase reaction is:

H2O2 + ZH2 ----peroxidase-->2 H2O + Z

That is, the enzyme converts hydrogen peroxide to water, obtaining the two hydrogen
atoms it needs for this from a “donor” molecule—called ZH2 in this example. Thus, at the same
time that hydrogen peroxide is being reduced, ZH2 is oxidized to Z. In this example, the letter
"Z" is not the symbol for a specific chemical; rather, it indicates that the enzyme can use several
different molecules as the source of the hydrogen atoms. For our experiments, we will use a
chemical that does not occur in plants, but that changes color—a fact that makes the reactions
very easy to monitor.

For example, horseradish peroxidase can use a variety of organic compounds as electron
donors and acceptors. Horseradish peroxidase has an accessible active site, and many compounds
can reach the site of the reaction.

For an enzyme such as cytochrome c peroxidase, the compounds that donate electrons are
very specific, because there is a much closed active site.

For many of these enzymes the optimal substrate is hydrogen peroxide, but others are
more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a
heme cofactor in their active sites, or a redox active cysteine or selenocysteine residues. The
nature of the electron donor is very dependent on the structure of the enzyme.

MATERIALS AND METHODS:

Refer to lab manual of biochemistry and metabolism

RESULTS:

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

Test Tubes

Time Optical Density, A

(seconds)

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

0 0.065 0.145 0.114 0.030 0.236

5 0.069 0.160 0.132 0.032 0.277

10 0.077 0.220 0.159 0.032 0.315

15 0.071 0.272 0.174 0.032 0.353

20 0.072 0.294 0.192 0.032 0.379

25 0.076 0.312 0.216 0.033 0.402

30 0.079 0.330 0.240 0.033 0.421

35 0.085 0.390 0.266 0.033 0.459

40 0.086 0.428 0.285 0.033 0.487

45 0.085 0.469 0.312 0.033 0.515

50 0.087 0.498 0.340 0.033 0.541

55 0.092 0.540 0.362 0.033 0.565

60 0.096 0.572 0.383 0.034 0.589

65 0.099 0.620 0.405 0.034 0.613

70 0.102 0.641 0.426 0.034 0.636

75 0.108 0.681 0.449 0.034 0.662

80 0.111 0.721 0.478 0.035 0.685

85 0.114 0.759 0.500 0.035 0.703

90 0.117 0.792 0.520 0.035 0.724

95 0.122 0.828 0.540 0.035 0.743

100 0.126 0.852 0.562 0.035 0.767

105 0.130 0.885 0.582 0.035 0.782

110 0.137 0.925 0.605 0.035 0.793

115 0.142 0.952 0.618 0.035 0.816

120 0.146 0.981 0.640 0.036 0.834

125 0.152 1.018 0.662 0.036 0.852

130 0.159 1.042 0.682 0.036 0.876

135 0.166 1.062 0.696 0.036 0.890

140 0.170 1.084 0.713 0.036 0.907

145 0.177 1.102 0.730 0.036 0.923

150 0.181 1.128 0.749 0.036 0.941

155 0.187 1.152 0.763 0.036 0.952

160 0.193 1.175 0.777 0.036 0.966

165 0.201 1.187 0.795 0.036 0.977

170 0.208 1.211 0.810 0.036 0.989

175 0.215 1.222 0.825 0.036 1.003

180 0.220 1.237 0.836 0.036 1.017

*Optical density does not have true unit; Absorbance, A is used.

Graph 1: Graph of Optical Density against Time

CALCULATIONS:

Zero order reaction rates:

Tube 1

= (0.068-0.025) / (180-0)s

=2.389×10-4 As-1

Tube 2
= (0.030-0.008)/(180-0)s

=1.222×10-4 As-1

Tube 3

= (0.012-0.003)/(180-0)s

=5.000×10-5 As-1

Tube 4

= (0.002-0)/(180-0)s

=1.111×10-5 As-1

Tube 5

= (0.044-0.027)/(180-0)s

=9.44×10-5 As-1

Enzyme peroxidase concentration in each tube:

Weight of dried crude peroxidase = 4.3mg

Volume of distilled water added = 10ml

Concentration of crude peroxidase =4.3mg/10ml

=0.43mg/ml

Calculated using M1V1=M2V2

Tube 1

= (0.43mg)(2ml)=(M2)(3.2)

=0.2687 mg/ml

Tube2

= (0.43mg)(1.5ml)=(M2)(3.2)
=0.2015 mg/ml

Tube 3

=(0.43mg)(0.5ml)=( M2)(3.2)

=0.0672 mg/ml

Tube 4

No reaction because there is no enzyme peroxidase to react with.

Tube 5

=(0.43mg)(1.0ml)=( M2)(3.2ml)

=0.1343 mg/ml

Concentration of commercial Peroxidase = 10mg/100ml

= 0.1mg/ml

Tubes Concentration of peroxidase, Zero order reaction rate, A/s

mg/ml
1 0.2687 2.389×10-4
2 0.2015 1.222×10-4
3 0.0672 5.000×10-5
5 0.1343 9.44×10-5

Table 2: The concentration of peroxidase and zero order reaction rate for different tubes.

DISCUSSION:

A zero-order reaction is a reaction that proceeds at a rate that is independent of reactant

concentration. In other words, increasing or decreasing the concentration of reactants will not
speed up or slow down the reaction, respectively. This means that the rate of the reaction is equal
to the rate constant, k, of that reaction.
 The purpose of adding the phosphate buffer to all the five tubes is to break the cells and
for reducing the thermal stability of crude peroxidase. Phosphate buffer accelerated the
denaturation process as well. P-phenylenediamine is the substrate for this experiment. The
peroxidase enzymes are used to degrade the hydrogen peroxide. Otherwise hydrogen peroxide
cannot be broken down without the enzymes. Once hydrogen peroxide is broken down, it will
give blue colour.

From the zero order reaction rates, we obtained the highest rate is tube 1 because it has
two-fold more peroxidase than tube 2 and tube 3. Second highest is tube 2 followed by tube 5
which has commercial peroxidase in it. Fourth is tube 3 and the lowest rate is tube 4. Tube 4 has
no added peroxidase. But however it still has some reaction because hydrogen peroxide can
react with phenylenediamine alone without enzymes. Tube 1 with 2ml of crude peroxidase has
highest zero order rate compared to tube 5 which used commercial peroxidase. Commercial
peroxidase is a pure form of peroxidase and it is better and more reactive and our crude
peroxidase. Supposedly, tube 5 should show the highest rate but it did not. This might be due to
only 1 ml of commercial peroxidase added to tube 5 whereas 2ml of crude peroxidase was
added to tube 1 which speeds the reaction. 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 o-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
their action rate.

The only possible errors that might have occurs must be only in tube 4. A little increase
in optical density obtained from the spectrophotometer indicated that there was still reaction of
hydrogen peroxide and p-phenylenediamine, even though lack with peroxidase. 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.

CONCLUSION:
As the concentration of peroxidase increases, the zero order reaction also increases. But if
compared with commercial peroxidase which is in pure form and our experimentally extracted
crude peroxidase, the reaction should be higher in commercial peroxidase.

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 March 2011, from
http://en.wikipedia.org/wiki/Hydrogen_peroxide
• Wikipedia 2009,Peroxidase, online, retrieved 25 March 2011, from
http://en.wikipedia.org/wiki/Peroxidase