BABS 2244 METABOLIC BIOCHEMISTRY

Name: Chong Khai En

Student ID: 15WAR08702
Group: RBS 2- Group 1
Date: 22-5-2015
Title: Experiment 1- Isolation and Properties of Peroxidase.
Introduction:
Peroxidase is an enzyme that acts as catalysts to allow a variety of biological
processes to take place. In fact, they promote the oxidation of many compounds using
naturally occurring peroxides, especially hydrogen peroxide (H2O2), which are reduced,
forming water. Peroxides are created as byproducts of various biochemical reactions
within organisms, but can cause damage as they are oxidizing agents. Peroxidases break
these compounds down in to harmless substances by adding hydrogen, obtained from
another molecule known as a donor molecule in a reduction-oxidation (redox)
reaction in which the peroxide is reduced to form water, and the other molecule is
oxidized. There are a large number of these enzymes, and they are found in plants and
animals, including humans.
Peroxidase is very large and complex molecules with complicated shapes and
multiple folds. Enzymes have an active site, which is the part of the molecule where the
reaction takes place. This may be in an easily accessible part of the molecule, or it may be
tucked away in a fold, where it can only be reached by a molecule of exactly the right
shape. Horseradish peroxidase (HRP) is an example of an enzyme that can use a wide
variety of donor molecules and peroxides.
Prosthetic groups are cofactors that bind tightly to proteins or enzymes and they
are not easily removed. Prosthetic groups can be as simple as a single metal ion bound
into the enzyme's structure, or may be a more complicated organic molecule, which
might also contain a metal ion. The enzymes carbonic anhydrase and catalase are simple
examples of the two types. The same cofactors can bind multiple different types of
enzymes and may bind some enzymes loosely, as a coenzyme, and others tightly, as a
prosthetic group. Some cofactors may always tightly bind their enzymes. These prosthetic
groups can also bind to proteins other than enzymes.

Aim:
1. To determine the effect of concentration of enzyme peroxidase on the rate of reaction
based on the optical density obtained through spectrophotometer.
2. To compare the rate of reaction catalyzed by crude peroxidase with that of the
commercial peroxidase.
3. To understand zero order reaction and its relationship to the theory of enzyme activity.

Method:
1. The peroxidase had been extracted from the raddish by the lab assistant and was stored
in a chilling condition.
2. Six test tubes were prepared and labeled based on the contents shown in Table 1 below.
Table 1: The contents and volumes of each test tube.
Tube

Enzyme
(ml)

Water
(ml)

Phosphate
buffer (ml)

pphenylenediamine
(ml)

Fluoride
(ml)

Commercia
l enzyme

0.1

0.1

0.5

1.5

0.1

0.1

0.1

0.1

3. 0.1 ml of hydrogen peroxide, H2O2 was added to the contents of each tube and was
shaken well.
4. Phosphate buffer was added to zero the absorbance. The change in absorbance at
450nm was recorded at every 5 seconds.
5. The optical density was recorded and the results were plotted in the graph.

Results:
Table 2: Optical density readings over time obtained from spectrophotometer on six test
tubes.
Optical Density, A
Time (s)
Tube 0
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
0

2.661

2.184

1.363

1.851

0.144

0.320

10

2.653

2.253

1.468

2.077

0.150

0.325

15

2.670

2.268

1.520

2.168

0.155

0.328

20

2.684

2.310

1.569

2.236

0.160

0.333

25

2.691

2.331

1.601

2.293

0.167

0.337

30

2.691

2.349

1.629

2.295

0.173

0.340

35

2.694

2.351

1.652

2.309

0.179

0.344

40

2.689

2.369

1.673

2.314

0.185

0.349

45

2.695

2.379

1.691

2.327

0.193

0.352

50

2.692

2.395

1.708

2.356

0.200

0.356

55

2.692

2.410

1.723

2.355

0.208

0.359

60

2.702

2.410

1.741

2.356

0.212

0.3633

65

2.669

2.420

1.752

2.374

0.219

0.367

70

2.679

2.423

1.771

2.371

0.226

0.370

75

2.692

2.433

1.780

2.374

0.231

0.374

80

2.693

2.428

1.789

2.386

0.238

0.377

85

2.686

2.445

1.801

2.386

0.244

0.381

90

2.688

2.450

1.811

2.395

0.250

0.385

95

2.703

2.445

1.822

2.397

0.256

0.390

100

2.687

2.448

1.830

2.400

0.261

0.393

Graph 1: Graph of optical density against time.

Graph of optical density against time

3.00

2.50

2.00

Tube 0density
Tube
1
Optical
(A)

1.50

Tube 2

Tube 3

Tube 4

Tube 5

1.00

0.50

0.00
0

10

20

30

40

50

60

Time (s)

Calculations:

70

80

90 100

2.661A
5s

Gradient of tube 0 =
= 0.5322 As-1
Zero order reaction rate = 0.5322 As-1

2.184A
5s

Gradient of tube 1 =
= 0.4368As-1
Zero order reaction rate = 0.4368 As-1

1.363A
5s

Gradient of tube 2 =
= 0.2726 As-1
Zero order reaction rate = 0.2726 As-1

1.851A
5s

Gradient of tube 3 =
= 0.3702As-1
Zero order reaction rate = 0.3702As-1

Gradient of tube 4 =

0.144A
5s

= 0.0288As-1

Zero order reaction rate = 0.0288 As-1

0.320A
5s

Gradient of tube 5 =
= 0.0640As-1
Zero order reaction rate = 0.0640As-1
Table 3: Volume of enzyme and zero order reaction rate of each tube.
Tubes
Volume of enzyme (ml)
Rate of reaction (s-1)
0

2.0

0.5322

1.0

0.4368

0.5

0.2726

1.0

0.3702

0.0288

0.0640

Graph 2: Graph of rate of reaction against volume of enzyme.

Graph of rate of reaction against volume of enzyme

0.6
0.5
0.4

Rate of reaction (As-1)

0.3
0.2
0.1
0
0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

Volume of enzyme (ml)

Discussion:
Enzymes are very efficient catalysts for biochemical reactions. They speed up
reactions by providing an alternative reaction pathway of lower activation energy. In
order to study the effect of increasing the enzyme concentration upon the reaction rate,
the substrate must be present in an excess amount; meanwhile the reaction must be
independent of the substrate concentration. Thus, the amount of product formed over a
specified period of time is dependent on the concentration of enzyme. These is said to be
zero order reaction because the rates are independent of substrate concentration, and are
equal to constant k.
From Graph 2, we know that the rate of reaction is directly proportional to the
concentration of enzyme present. As we compared the zero order reaction rates of Tubes
0, 1 and 2, we found that when the concentration of enzyme increases, the reaction rate
also increases. In another word, there are more enzymes available for successful collide
with the substrate molecules and bond together when the concentration of enzyme is

increased. Subsequently, more products are formed at a shorter period of time because
substrates have been used up faster.
Some substances reduce or stop the catalytic activity of enzymes in biochemical
reactions by blocking or distorting the active site of enzyme. These chemicals are called
inhibitors. Inhibitors that occupy the active site and prevent a substrate molecule from
binding to the enzyme are called competitive inhibitor. Another type is non-competitive
inhibitor, which attach to other parts of the enzyme molecule to distort its shape. When
we compared Tubes 1 and 3, even they have the same concentration of enzyme, but
reaction rate of Tube 3 is lower than Tube 1 due to the presence of fluoride as an enzyme
inhibitor. Fluoride switches off the enzyme by attacking its weakest link which is the fine
balanced network of hydrogen bonds surrounding the enzymes active site. Fluoride
inhibits enzymes by preventing these natural catalysts carrying out their essential tasks.
The crystal structure of a fluoride-inhibited peroxidase enzyme shows that the fluoride
ion attaches itself to the iron atom at the heart of the enzyme and then disrupts the active
site by attracting groups that can form strong hydrogen bond to it. Thus, this inactivates
the enzyme by changing its shape or molecular formation.
We can see that in Tube 4, there is little increase in optical density obtained from
the spectrophotometer. This indicates that there was still has reaction of hydrogen
peroxide and p-phenylenediamine even though lack of peroxidase. It is maybe due to the
contamination of little amount of inorganic catalyst in water or any other solutions being
added together into the test tube. The inorganic catalyst which present in little amount can
be metallic ions such as copper ion, manganese dioxide and silver ion. Thus, the reaction
may possibly due to catalysis by trace amounts of these metal ions.
Other than that, we compared the reaction rate of using crude enzyme and
commercial enzyme; obviously we found that the reaction rate of using crude enzyme is
much higher than the one with commercial enzyme. Tubes 0, 1, 2, 3 have higher rate of
reaction than Tube 5 which contained commercial enzyme. One possible explanation for
this result was the impurities of commercial enzyme preparation of peroxidase. Although
the concentration of commercial enzyme in Tube 5 is higher than concentration of crude
enzyme in Tubes 1, 2, 3, however, the commercial enzyme might contain largely of other
compounds presence in it and only little amount of peroxidase. In addition, the
preparation techniques in commercial industry were much sophisticated and cause more
mistakes.

Conclusion:
Zero order reaction rate is independent of substrate concentration, and is said
equal to constant k. In order to study the effect of increasing the enzyme concentration
upon the reaction rate, the substrate must be present in an excess amount; meanwhile the

reaction must be independent of the substrate concentration. Thus, the amount of product
formed over a specified period of time is dependent on the concentration of enzyme. In
this experiment, fluoride acts as an enzyme inhibitor. Fluoride attacks peroxidases
weakest link which is the fine balanced network of hydrogen bonds surrounding the
enzymes active site. Fluoride inhibits enzymes by preventing these natural catalysts
carrying out their essential tasks. It also inactivates the enzyme by changing its shape or
molecular formation.
References:
1. Biochem Enzyme, published by Peter Hong Leong Cheah, viewed on 1st June 2015,
<http://www.scribd.com/doc/18018345/Biochem-Enzyme#scribd>
2. ALevelNotes.com, viewed on 1st June 2015,
<http://alevelnotes.com/factors-affecting-enzyme-activity/146>
3. Enzymes, viewed on 1st June 2015,
<http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm>
4. Commercial Production of Enzymes, written by Aritri Ghosh, viewed on 2nd June 2015,
<http://www.biotecharticles.com/Biotech-Research-Article/Commercial-Production-ofEnzymes-873.html>
5. Coenzymes, Cofactors & Prosthetic Groups: Function and Interactions, viewed on 2nd
June 2015,
<http://study.com/academy/lesson/coenzymes-cofactors-prosthetic-groups-function-andinteractions.html>