Experiment 8: Intracellular Location of Respiratory Enzymes

Group: ​RBS 2 Group 1

Names: ​Chin Jia Yue (20WLR08551) Loo Jia Ying (20WLR08567)

Kiew Wei Yong (20WLR08560) Low Ting Yue (20WLR08568)
Lee Ka Man (20WLR08563) Ng Si Qi (20WLR08569)
Lim Chen Yee (20WLR08565) Wong Pui Mun (20WLR08576)
Liow Khai Li (20WLR08566)

Part A: The reduction of methylene blue by a rat liver homogenate

Discussion
Methylene blue is widely used as a redox indicator in analytical chemistry. This solution is blue
when in an oxidizing environment, but will turn colorless if exposed to a reducing agent.
Oxygen-rich environments are said to be "oxidizing." Oxidizing chemicals, such as oxygen and
chlorine, strip electrons away from other atoms. Methylene blue indicates the presence of
oxidizing agents because it is oxidized by these compounds. When electrons are stripped from
methylene blue, the resulting molecule imparts a blue color to the solution. Under aerobic
reaction conditions, molecular oxygen, present in the reaction solution, rapidly oxidizes MBH to
M B+ .

Oxygen molecules were consumed in the cellular respiration. Thus for the sample that respiration
occurs, methylene blue will be decolorized when it receives an electron from NADH. NADH is
generated in glycolysis which is necessary for aerobic respiration. Due to this condition. Sample
1 that turned lighter blue was believed that cellular respiration has occurred. In sample 2 that
contained boiled homogenate and methylene blue, only the bottom part of the sample was turned
lighter. High temperature in boiling caused the respiratory enzymes in the liver to be denatured.
No decolourized should be expected in this sample. However the methylene blue in the bottom
portion of this sample was reduced. This might be due to the incomplete boiling of the
homogenate which caused the enzymes to be not fully denatured. Thus the enzymes still can
function to carry out respiration and reduce the methylene blue. Sample 3 contained only
distilled water and methylene blue. No homogenate was added to consume the oxygen in the
water. Therefore the methylene blue remained deep blue colour in the oxygen-rich water.

Questions
1. Does complete decolorization occur in any tube? If not - why not?
No complete decolorization occurs because a complete decolorization means the blue colour of
solution turned into fully colourless. However, there is no tube with colourless. For tube 1, the
blue colour of solution just turned lighter. It might be due to the amount of enzyme is not enough
to reduce all the methylene blue. For tube 2, it should not have any colour changes. However, the
bottom of the tube is slightly lighter which indicated the incomplete boiling. The remaining
enzyme could still reduce the methylene blue. For tube 3, no enzyme produces at all, therefore
no decolorization.

2. What substances in the homogenate can be causing the reduction of methylene

blue?
NADH produced by the enzyme will reduce methylene blue by donating one electron to it.

3. Is the use of mineral oil an ideal way of excluding atmospheric oxygen?

Yes, mineral oil can reduce the penetration of atmospheric oxygen better compared to other oils
such as vegetable oils. This helps to prevent atmospheric oxygen from entering which will affect
the results obtained. Oxygen is able to diffuse through the oil layer, but the diffusion rate will be
very slow.

4. Shake each tube vigorously and note the decolorization of methylene blue
Shaking test tube 1 and 2 causes a change in the blue color, which turns to the initial dark blue
color. When methylene blue is reduced, it will turn light blue to colorless, depending on the
amount of reducing agent. When oxidized, it turns back to the blue color.
Shaking test tube 3 will not have any changes.

Part C: The location of succinic dehydrogenase

Discussion
Table 2 Observations of the succinic dehydrogenase activity
Tube no. Observations

1 Colour remain unchanged

2 Colour remain unchanged

3 Blue colour decolourised, become lighter

4 Blue colour decolourised, become lighter

In tube 1 and tube 2, which both contained supernatant, the solutions were in oxidised form of
methylene blue as they were blue in colour. Their colour remained unchanged after the test. In
tube 3 and 4, which both with particulate, the solutions decolourised, there were in the reduced
form of methylene blue.

Succinate dehydrogenase is the only enzyme of the TCA cycle that is also part of the electron
transport system, thus, it is located in the inner membrane. Succinate dehydrogenase and its
coenzyme flavin adenine dinucleotide (FAD), represented as the complex E-FAD, oxidize the
metabolite succinate to fumarate. Succinate dehydrogenase removes electrons from succinate,
which reduces FAD, thus reducing the enzyme complex to E-FADH​2​. The reduced coenzyme
then transfers the electrons to coenzyme Q to be taken through the rest of the electron transport
chain. All the enzymes involved in the TCA cycle were extracted in the cell-free state. It was
noticed that succinate dehydrogenase activity was not shown in supernatant but particulate
fraction. This means that the succinic dehydrogenase is inside the particulate fraction.
Part D: The location of cytochrome oxidase
Discussion

Tube 0.5 mL of 0.5 mL 0.5 mL KCN 0.5 mL H2O Colour

particle supernatant change
suspension

1 + - - + Turn dark
brown

2 - + - + Turn light
brown

As shown in the figure 3(i) and 3(ii), the color of the tubes changed after incubation. The color of
tube 1 that was provided with particulate change from white with slightly precipitate to dark
brown as well as the colour of tube 2 which contained the supernatant changed from light pink to
light brown.

Cytochrome oxidase that is also known as complex IV mitochondrial respiratory chain plays a
fundamental role in energy production of aerobic cells. This multimeric enzyme of the inner
mitochondrial membrane catalyzes the last step of respiration. ​The cytochrome oxidase is located
at the inner membrane of the mitochondria. The enzyme cytochrome c oxidase is a larger
transmembrane protein complex found in bacteria and the mitochondria of eukaryotes. The
cytochrome c received the electron from another complex. It converts the oxygen molecules with
the proton to water. At the same time, 2 protons are pumped out to give energy in the form of
ATP to the reaction. In the last steps of the energy production process, cytochrome oxidase
oxidizes the waste products from the end of the energy making process, converting reactive
species, H+ and dioxygen (O​2​), to a more stable molecule, water (H​2​O).

Questions
1. In which fractions is cytochrome oxidase localised?
The fraction that cytochrome oxidase localised is the particulate fraction (microsome).

2. Explain the effect of CN .

When cyanide binds to the cytochrome oxidase, it inhibits the protein from functioning and
leads to the chemical ​asphyxiation of cells. Higher concentrations of molecular oxygen are
needed to compensate for increasing inhibitor concentrations, leading to an overall reduction in
metabolic activity in the cell in the presence of an inhibitor. In short, cyanide can inhibit
cytochrome oxidase by binding to regulatory sites on the enzyme thus reducing the rate of
cellular respiration.

Part E: A comparison of lactate and succinate dehydrogenases

Discussion​ cy
Based on the results obtained figure 4(i), before the reaction all the test tubes show the same intensity of
colour. After reaction, all the test tubes show variation in their intensity of colour. Based on the results
constructed in figure 4(ii), test tube 4, 8 and 10 show very blue solution. This is because in test tube 4
there is absent of succinate, but it contains lactate. The oxidative decarboxylation is not too effective in
lactate thus, the solution remains its intensity. In test tube 8, although there is the presence of succinate,
but FAD+ is absent. In the absent of FAD+, the oxidative decarboxylation will not be able to conduct
efficiently. In test tube 10, there is also contains succinate with the absences of FAD+. In this case, the
water added is less than test tube 8, so the intensity of blue colour solution is less intense than test tube 8.
In test tube 5, the intensity of solution is blue which is lighter than test tube 4, 8 and 10. This is because
the presence of FAD+ and pyruvate. Succinate dehydrogenase is FAD+ dependent. In test tube 9, the
intensity of the blue colour solution reduced which turns into light blue. This is because the presence of
FAD+. Succinate dehydrogenase is FAD+ dependent. In test tube 1, 2 and 6, there are very light blue
solution. This is because in both test tube 1 and 2, all the contents are the same except the volume of
water. The volume of water in test tube 1 is lesser than test tube 2, so the intensity of blue colour solution
will be more intense than test tube 2. In test tube 6, there is the present of FAD+ which is need for
succinate dehydrogenase. Thus, the intensity of blue colour solution is lighter. In test tube 3, 7 and 11, the
solution is clear. This is because in test tube 3, there is the present of FAD+ and the succinate
dehydrogenase is FAD+ dependent. In both test tube 10 and 11. There are the present of succinate and
FAD+. Therefore, the reaction will be more efficient than the test tube that contains lactate. However, the
test tube 11 will appear with very little blue solution. This is because the volume of water added in test
tube 11 is lesser than test tube 10. Thus, the reaction rate reduced.

Questions
3. Compare the requirements of lactate and succinate dehydrogenases for NAD+. Explain
the observed effects of pyruvate and malonate on these two enzymes.
The lactate dehydrogenases need the NAD+ to change the colour to dark blue while the succinate
dehydrogenase does not need the NAD+ to change the colour. Lactate dehydrogenase help to
regenerate the NAD+ while the succinate dehydrogenase helps to produce the NADH. When
adding the pyruvate to the lactate, the solution turns to dark blue. Whereas, for succinate, the
solution gives a very light blue colour. When adding the malonate to the lactate, the solution is in
a very light blue colour. This is because the pyruvate in the lactate dehydrogenase produced the
lactic acid. The malonate inhibited the reaction of lactate dehydrogenase.

4. What do you think would be the effect of adding cyanide to tube (5)?
The solution does not change in colour. This is because the cyanide is a toxin that can block the
reduction reaction.