FACULTY OF APPLIED SCIENCES AND

COMPUTING (FASC)
KUALA LUMPUR CAMPUS
BACHELOR OF SCIENCE (HONS) IN
BIOSCIENCE WITH CHEMISTRY

BABS2244 METABOLIC BIOCHEMISTRY

EXPERIMENT 15
Redox Enzymes
NAME

ID NO.

Amirah Chan binti Latif

15WAR08520

Chey Sze Ying

15WAR09194

Chong Khai En

15WAR08702

Goh Yee En

15WAR08280

PROG/GROUP

RBS2 Group
1

Introduction
:
Biochemical reactions in living organisms are essentially energy transfers.
In biological systems, oxidation is often coincident with the loss of
hydrogen, dehydrogenation. The enzymes that catalyze these oxidations
are called dehydrogenases. Oxidation-reduction reactions (Redox
reactions) must occur together. Electrons are transferred from the
reducing agent to the oxidizing agent such that the reducing agent is
oxidized and the oxidizing agent is reduced. It is convenient however to
describe the electron transfer reaction as two half reactions, one for the
oxidation of the reduced species and one for the reduction of the oxygen
species. Such coupled reactions are referred to as redox reactions. The
metabolic processes glycolysis, Kreb's Cycle, and Electron Transport
Phosphorylation involve the transfer of electrons (at varying energy
states) by redox reactions.

Figure 1. Passage of electrons from compound A to compound B.

(When A loses its electrons it is oxidized; when B gains the electrons it is
reduced.)

Figure 2. Oxidation/reduction via an intermediary (energy carrier)

compound, NAD+.

Electrons are transferred from one molecule to another in four different

ways:
1 Directly as electrons. (Fe2+ + Cu2+ Fe3+ + Cu+)
2 As Hydrogen atoms. The hydrogen atom contains a proton, H + and
an electron, eAH2 A + 2e- + 2H+
3 As a Hydride ion, H-. The hydride ion has two electrons and is highly
reactive. In biological systems it is directly transferred to NAD-linked
dehydrogenases.
4 Through the direct combination with oxygen. Molecular oxygen
combines with organic reactants to oxidize hydrocarbons to
alcohols, aldehydes to acids.
RCH3 + O2 RCH2OH

Aim :
1. To determine the presence of enzymes in various plant and animal
tissues.
2. To compare the rate of enzyme activity between fresh and boiled
samples.

DEHYDROGENASE ACTION
Method

Part A
A series of 5 test-tubes labeled A to E were prepared. 5 ml of 0.05%
methylene blue solution was added in water to each. The lean meal with
equal small pieces that had prepared by lab assistant was treated as
indicated below:
Samples

Contents

Test-tube A

2 pieces of meat, freshly cut.

Test-tube B

2 pieces of meat, previously boiled and cooled.

Test-tube C

2 pieces of meat, previously minced.

Test-tube D

2 pieced of meat, thoroughly washed under running tap

water.

Test-tube E

Control.

After that, all 5 test-tubes were corked and left undisturbed in a water
bath maintained at a constant temperature of 37 OC. The tubes were not
shaken once the contents had mixed. The contents were observed against
a white background and any changes in the 5 tubes were recorded at
intervals of 5 minutes; starting 4, 5, 10, 15 minutes until two
subsequent observations show no change in color, intensity and
distribution. The test tubes were uncorked and the contents were shaken
thoroughly. All observations and the conclusions were recorded in a
tabular form.
Part B

A duplica a series of 3 test-tubes containing 5 ml of 0.05% methylene blue

was prepared and labeled them Fa, Fb, Ga, Gb, Ha and Hb. The following
fresh specimens were added to the a series and an equal quantity of the
same specimen previously boiled and cooled was added to the b series
as shown in table below:
Samples

Contents

Test-tube F

2 pieces of liver, freshly cut (each piece about 5mm

cube)

Test-tube G

2 pieces of potato freshly cut (about 5mm cube)

Test-tube H

2 ml yeast suspension (10% yeast solution).

All the 3 pairs of test-tubes were incubated in a water bath at 37 OC. Again,
the contents were observed against a white background and any changes
in the 5 tubes were recorded at intervals of 5 minutes; starting 4, 5, 10,
15 minutes until two subsequent observations show no change in
colour, intensity and distribution. The test tubes were uncorked and the
contents were shaken thoroughly. All observations and the conclusion we
made were recorded in tabular form.
Results

Observation at 5th minute

Observation at 10th minute

Observation at 15th minute

Observation at 20th minute

Observation at 25th minute

Observation at 30th minute

Observation at 35th minute (final)

Part A
Samples
Test-tube A

Observations

Deduction

Solution changes from

blue to greenish-blue.

-Dehydrogenase is present.

Presence of a
transparent layer
around the meat

Test-tube B

Test-tube C

Test-tube D

Solution remains blue

-Some dehydrogenase is
still present maybe
Transparent layer at the
because meat was not
base of test tube,
boiled properly.
around the meat is
formed

Solution changes from

blue to light blue

-Dehydrogenase is present,
but the amount is less
than test-tube A.

Solution remains blue

-Absence of
dehydrogenase.

Greenish-blue
transparent layer at the
base of test tube,
around the meat is
formed

Test-tube E

Color of solution
remains blue.

-Absence of
dehydrogenase.

Part B
Samples
Test-tube Fa

Test-tube Fb

Observations

Deduction

Solution changes from

blue to greenish-blue.

-Dehydrogenase is present.

Solution is slightly
cloudy

Color of solution
remains blue.

A yellowish layer can be -The liver was not boiled

observed around the
properly.
boiled liver.

-Dehydrogenase enzymes
are slightly present.

Test-tube Ga

Color of solution
remains blue.

- Dehydrogenase is
absent.

Color of solution
remains blue.

- Dehydrogenase is
absent.

Solution turns cloudy,

greenish-blue color

Test-tube Gb

Test-tube Ha

-The yeast is stale, thus we

cannot know that
whether the yeast
contains dehydrogenase
or not.

Test-tube Hb

Discussion

Solution turns cloudy,

greenish-blue color

-The yeast is stale, thus we

cannot know that
whether the yeast
contains dehydrogenase
or not.

1. In what ways have the different treatments on the meat

affected their action on methylene blue? Suggest possible
explanations.
One of the meats is being boiled completely while the other meat is
not. Dehydrogenases are present in meats. They initiate the oxidation
process by transferring hydrogen from a substrate to a hydrogen
acceptor. Methylene blue solution is often used as an artificial
hydrogen acceptor. A completely boiled meat will have no
dehydrogenases because they are denatured under high temperature.
Thus, no hydrogen is present to be accepted by the methylene blue

solution and its color remains unchanged. On the other hand, an

unboiled meat has dehydrogenases. Hence, the dehydrogenases will
transfer the hydrogen to the methylene blue solution and reduction
occurs. When reduced, methylene blue solution will change from blue
to colorless. The decolorization of methylene blue solution acts as a
visible indicator of the presence of dehydrogenases in meat tissue. The
degree of dehydrogenases activity affects the rate of decolorization of
methylene blue solution.

2. Explain the significance of setting up test-tube E.

Test tube E is used as a control in the experiment. There is nothing to
be added inside this test tube which means there will be no reaction
in this test tube. Besides, the color of methylene blue in test tube E
is unchanged. Thus, we can compare the color of methylene blue in
test tubes A to D with test tube E which its methylene blue color
will never being changed. The color change in test tubes A to D
indicate that there is reaction in the test tubes.

3. What is the purpose of setting up tubes B, Fb, Gb and Hb?

These tubes are being set up for comparison of tubes A, Fa, Ga and Ha
respectively. The tissues used in these test tubes are being
completely boiled and cooled. Thus, the hydrogenases will be
denatured and there is no reaction inside these tubes. The color of
methylene blue solution will remain unchanged since no hydrogen is
present. So, we can observe whether there is reaction in the tubes A,
Fa, Ga and Ha very clearly by comparing the color of tubes B, Fb, Gb
and Hb.

4. What does shaking the contents indicate about the reaction of

the various mixtures?
Shaking the contents is important so that the tissues and the solution
can mix well. The contents and the solution are being mixed well to
ensure that there is a complete reaction in the tubes. Random error
can be avoided and we can get more accurate observations.

5. Point out similarities or differences in the effect of the

different tissues on methylene blue.
For all the boiled tissues in this experiment including liver, meat and
potato, there will be no reaction when these boiled tissues are being
added to the methylene blue solution. This is because methylene blue
solution which is the artificial hydrogen acceptor cannot accept
hydrogen which is absent in these boiled tissues. Since there is no
hydrogen to be received by methylene blue solution, the methylene
blue solution will remain unchanged which is blue color. On the other
hand, for the same tissues such as boiled meat and unboiled meat,
they will be put into the test tubes with same solution. After 25 30
minutes, we can observe that the in test tube of boiled meat, the
methylene blue solution turns to colorless but in the test tube of
unboiled meat, the methylene blue solution remains blue. This
indicates that the hydrogenases in boiled meat are denatured while
the hydrogenases in unboiled meat are not being denatured.

6. What correlation can you draw about the activity and the
source of the enzyme?
Different source will contain different amount of enzyme. The enzyme
will speed up the reaction by lower the activation energy. The rate of
the reaction depends on the presence of enzyme. If there are more
enzymes, the reaction will be faster compared to the reaction with
fewer enzymes.

CATALASE ACTION
Method

A duplicate series of 5 test-tubes labeled Aa, Ab, Ba, BbEa, Eb were

prepared. Then, 5 ml of hydrogen peroxide (10 volumes) was added to
each test tube. The following fresh specimens as shown in table below
were added to the a series and a similar quantity of the same specimens
previously boiled for 5 minutes and cooled was added to the b series:
Samples

Contents

Test-tube A

2 pieces of meat (each 5mm cube).

Test-tube B

2 pieces of liver (each 5mm cube).

Test-tube C

2 pieces of potato (each 5mm cube).

Test-tube D

2 pieces of apple (each 5mm cube).

Test-tube E

2 ml of yeast suspension.

Test-tube F

A piece of plant stem/ leaf cut out of the entire organ

with a sharp blade so that it presents both the intact
surface and cut cells.

Test-tube G

A sample of potato/ meat plus 95% alcohol to cover.

Allow to stand for 5 minutes. Pour out the alcohol.

Test-tube H

A pinch of manganese dioxide.

The frequency and size of escaping bubbles were observed for each test
tube. The observations and conclusions for each test tube were tabulated.

Results

Samples

Observations

Deduction

- Catalase is present.

Test-tube Aa

Effervescence could be
observed when the
meat is dropped in.

Test-tube Ab

- Catalase is present in
Effervescence occurs
slowly when boiled meat small quantity.
was dropped in

Test-tube Ba

Effervescence occurs
very rapidly when the
liver was dropped in

- Catalase is present in
large amount.

Test-tube Bb

No bubbles formed

- Catalase is absent
because the liver had
been boiled.

Test-tube Ca

Few noticeable bubbles

formed.

- Catalase is present in
small quantity.

Test-tube Cb

Few noticeable bubbles

formed.

- Catalase is present in
small quantity.

Test-tube Da

Test-tube Db

Test-tube Ea

Very few bubbles

formed.

- Catalase is present in
small quantity.

Very few bubbles

formed.

- Catalase is present in
small quantity.

No effervescence
occurred.

- Catalase is present in
yeast, but in this case, the
yeast is stale, thus the
result was affected.

Test-tube Eb

Test-tube F

Test-tube G

No effervescence
occurred.

- Catalase is present in
yeast, but in this case, the
yeast is stale, thus the
result was affected.

Bubbles formed around

the edges of the leaf.

- Catalase is present in the

plant tissues.

Effervescence occurs
rapidly.

- Catalase is present.

Test-tube H

Very little effervescence. - Catalase is present.

- Manganese dioxide is an
inorganic catalyst (but not
an enzyme) for the
breaking down of
hydrogen peroxide.

Discussion

1. What is the effect of boiling on the different plant and animal

tissues?
One of the factors that causes enzyme to be destroyed is the exposure
of the enzymes to heat. When boiling is carried out, excessive heat is
supplied to the plant and animal tissues, causing the cells to be killed
and enzymes are eventually denatured.

2. Why is it necessary to set up the b series of test tubes?

The b series of test tubes allows us to observe whether catalase in
these boiled tissues is able to catalyze the decomposition of hydrogen
peroxide. Therefore we can compare its rate of enzyme activity with its
respective fresh tissues.

3. Does boiling affect the action of manganese dioxide? Why?

Boiling is able to affect the action of manganese dioxide, but the
effects are not too noticeable. This is because manganese dioxide is an
inorganic catalyst and inorganic catalysts are less sensitive to high
temperatures. Therefore, effervescence could be observed in test-tube
G.

4. Is there any difference in the amount of gas evolved when

different tissues are used? What conclusions can you draw
regarding the activity of the enzyme when it is of different
tissue origin?
Based on the observations, more gas is evolved when an animal tissue
is used compared to plant tissues, whereby the fresh liver shows the
highest amount of gas evolved. Catalase, which catalyzes the
decomposition of hydrogen peroxide into oxygen and water (resulting
in an effervescence), exhibits a higher activity in animal tissues as
animals have a greater need for metabolism and cellular respiration. It
functions as a protective enzyme and allows living organisms to keep
living, which is crucial especially in animals.

Conclusion
:
Hydrogenases are commonly present in plant and animals tissues. In this
experiment, its function is to transfer hydrogen from the substrate which
is the tissues to the hydrogen acceptor which is the methylene blue
solution. The methylene blue solution will be reduced when hydrogen is
being accepted and the color will change from blue to color. The color
change of methylene blue solution indicates that there is reaction.
Besides, the rate of enzyme activity for fresh tissues is faster than the
rate of enzyme activity for boiled tissues. This is due to the hydrogenases
in boiled tissues is denatured under high temperature while hydrogenases
in fresh tissues is sufficient for reaction to take place. Thus, the rate of
enzyme activity for boiled tissues will be slower.

References

1. Rastogi, VB 1997, Modern biology, Pitambar Publishing Company (P)

Ltd., New Delhi.

2. Diffen 2015, Catalyst vs enzyme,

<http://www.diffen.
com/difference/Catalyst_vs_Enzyme>

viewed

June

2015,

3. MadSci Network 1998, Re: Catalase activity in various cells, viewed 8

June
2015,
<http://www.madsci.org/posts/archives/199811/910885187.Bc.r.html>
4. Catalase by Jennifer McDowall, viewed on 12th June 2015,
<http://www.ebi.ac.uk/
interpro/potm/2004_9/Page1.htm>
5. Methylene Blue Reductase Test, viewed on 12th June 2015,
<http://vlab.amrita
.edu/?sub=3&brch=73&sim=1630&cnt=1>