Results

Table 1: Observations for Each Test Tubes of Na Succinate at Different Time Intervals for
Methylene Blue Experiment.
Observation
Time Interval (mins) 15 30 60
Test Tube A Dark purple intensity Dark purple intensity Dark purple intensity
Test Tube B Dark purple intensity Light blue intensity Pale blue
Test Tube C Dark purple intensity Dark blue intensity Light Blue

Table 2: Observations for Triphenyl Tetrazolium Chloride Salt Experiment with Their
Respective Absorbance Reading at 490nm.
Test Tubes 0 mins After 20 mins During 60 mis After 60 mins Absorbance
(O.D) @
490 nm
 1 1
2 1
3 1
4 1
5 1
4 4
2 2
3 3
1 1
2 2
4 0.383
1 0.106
3 0.414
1 0.162
2 0.247

Discussion

In part, A of the experiment, the reagents used were 0.4 M Na Succinate, an enzyme from
Yeast, 0.4 % methylene blue, and 0.2 M Na-iodoacetate and oil. Na Succinate is the substrate for
the yeast enzyme. Na succinate will bind to the active site of the yeast enzyme which catalyzes
the oxidation of Na succinate. Methylene Blue on the other hand is a blue dye in its oxidized
state but becomes colourless in the reduced state. This property of Methylene Blue allows it to
measure the activity of enzymes (dehydrogenases). Since methylene blue has a higher affinity for
hydrogen atoms, some hydrogen atoms are passed from reduced FAD (hydrogen carrier) to
methylene blue. This causes the reduction of methylene blue leading to the disappearance of the
blue colour in the experiment. Oil was used in the experiment to prevent oxidation of Methylene
Blue. Na iodoacetate however is a competitive inhibitor for the yeast enzyme (Succinate
dehydrogenase). Competitive enzyme inhibitors possess a similar shape to that of the substrate
molecule and compete with the substrate for the active site of the enzyme. This prevents the
formation of enzyme-substrate complexes. Therefore, fewer substrate molecules can bind to the
enzymes, so the reaction rate is decreased. So, in the experiment with the addition of Na-
iodoacetate, we would expect that the methylene blue will be more intense since the reaction
enzyme has been inhibited and hence the reaction will be slowed. In test tube A, only 0.4 M Na
Succinate and 0.4 % methylene blue were added and there was a constant dark purple intensity at
each interval as expected since there were no enzymes to catalyze the reaction. Test Tube B on
the other had the enzyme, and the colour intensity decreased from dark purple to pale blue as
expected. The solution in test tube B changed intensity because as the reaction proceeded
Hydrogen was passed to Methylene Blue causing reduction and leading to the disappearance of
the blue colour in the experiment. Similarly, Test tube C decreased in intensity. However, the
intensity was higher than in the test tube in B (light blue) as expected which is due to the
presence of Na-iodoacetate which inhibited the enzyme from functioning optimally so the
amount of Hydrogen getting transferred to methylene blue for reduction was lowered.
The reagents used in part B of the experiment were 0.1 M NaH2 PO4 pH7.4 , 0.5 M ἀ-
glycerol phosphate ,0.5 M lactate , 0.5% TTC (Triphenyl tetrazolium chloride), 0.5% NAD and
yeast suspension . The 0.1 M NaH2 PO4 buffer was used to maintain a constant pH of 7.4. The ἀ-
glycerol phosphate and lactate are the substrates that different enzymes in the yeast suspension
will bind to and catalyze their oxidation. An indicator in the form of Triphenyl tetrazolium
chloride was used for measuring the enzymatic activity of the enzymes in the yeast suspension.
Triphenyl tetrazolium chloride is a redox indicator that is colourless in its oxidized state and
turns red when it is reduced, it can be reduced by hydrogen carriers such as NAD and become
red formazan as seen in the experiment. The NAD used in the experiment is a coenzyme that
mediates redox reactions through a transfer of electrons and hydrogen between NAD and
Triphenyl tetrazolium chloride. This transfer of Hydrogen to Triphenyl tetrazolium chloride will
reduce the indicator as stated to red Formazan. Other reagents in the experiment are methanol
and acid were used to remove any water-soluble things in the solution left. The hexane extracts
the formazan and makes it show more in the solution During the experiment, only the buffer,
TTC and yeast suspension were added to test tube 1and a high red intensity was observed as
expected with an absorbance of (0.383 O.D ). A reaction occurred in tube 1 because lactate and
ἀ-glycerol phosphate (substates) are also present in the yeast suspension with their respective
enzymes for catalyzation and leading to the reduction of Triphenyl tetrazolium chloride which
accounted for the colour (Red) development. However, in test tubes 2 and 3 the buffer, TTC, ἀ-
glycerol phosphate and yeast suspension were added with NAD also being added to test tube 3.
Test tube 3 was observed to have a higher intensity than tube 2 because tube 3 contained added
NAD which would accept more Hydrogen from the substrate (basically increasing the activity of
the dehydrogenase) and transfer it to TTC reducing it to insoluble red formazan. On the other
 Questions

1. What happens to the colour in the test tube (for part A)? Explain

In test tube A, methylene blue, Na – succinate, water and oil droplets were added. The
solution was observed to be dark purple, similar in colour to methylene blue. Methylene blue
was unable to get reduced due to the absence of enzymes in the reaction. A pale blue colour was
observed in test tube B, this is because when compared to test tube A, methylene blue, oil
droplets, sodium succinate, water, and enzyme were all present in B. Additionally there was no
inhibitor so methylene blue could now be reduced by accepting Hydrogen and started to
decolourized as the enzyme performed its function. Test tube C contained all the components of
tube b however it also contained the inhibitor Na- iodoacetate. The colour observed in c is
slightly darker than B (light blue) which is due to the inhibitor Na- Succinate being present. The
enzyme was able to carry out its function at one point but once Na-iodoacetate was added its
activity reduce and hence the reduction of methylene blue stopped (remained the colour it is at
that point).

2. How is the enzyme affected by Na-iodoacetate?

Na-iodoacetate is a competitive inhibitor of the enzyme, therefore, it has a similar shape

to that of the substrate molecule and competes with the substrate for the active site of the
enzyme. This prevents the formation of enzyme-substrate complexes and reduces the activity of
the enzyme.

3. Which enzymes seem to be present in the yeast as determined by this experiment?

Due to the reduction in TTC to mono formazan, we can conclude that NADH was formed
to reduce the TTC, so, therefore, we can infer the class of enzyme in the yeast is dehydrogenase
and the name of the two enzymes determined were lactate dehydrogenase and ἀ-glycerol
phosphate dehydrogenase.

4. How can you account for the development colour in test tube 1?

Test tube 1 in the experiment consisted of the buffer, Triphenyl tetrazolium chloride and
yeast suspension. A reaction occurred because lactate and ἀ-glycerol phosphate (substates) are
also present in the yeast suspension with their respective enzymes for catalyzation and leading to
the reduction of Triphenyl tetrazolium chloride which accounted for the colour (Red)
development.

5. What is the role of NAD+? Does any activity occur in the absence of the co-enzymes?

The role of the coenzyme NAD+ is to help the enzyme to catalyze the reaction by serving
as a hydrogen acceptor and getting reduced to NADH in the process. It makes the redox reaction
easier by facilitating the transport of hydrogen and electrons. During the experiment, the role of
NAD+ is to accept and transfer electrons and hydrogen to TTC, which reduces TTC to the Red
insoluble Formazan.
 No activity is expected to occur in the absence of the NAD+ co-enzyme, however,
activity did occur in tubes 1, 2 and 4 where no NAD+ was supplied, but there was a pink colour
to indicate that TTC was reduced. This suggests that the yeast suspension may already have the
enzyme which allowed enzyme activity to occur indicated by the pink colour.

6. What common function may be ascribed to each of these enzymes in intermediary

Metabolism?
Both enzymes lactate dehydrogenase and ἀ-glycerol phosphate dehydrogenase are enzymes that
catalyze reduction reactions through the transfer of hydrogen ions (protons) from the substrate to
an acceptor or co-enzyme. During both reactions, NAD was reduced to NADH, so therefore the
common function of these enzymes in the intermediary metabolism is to reduce NAD to NADH
via hydrogen transfer.

7. Using charts of metabolic pathways, try to decide what the significance of alpha-
glycerol phosphate dehydrogenase might be in terms of the interaction of
carbohydrates and lipid metabolism.

Glycerol-3-phosphate dehydrogenase (GPDH) is an enzyme reversibly catalyzing the reduction

of dihydroxyacetone phosphate (DHAP) from the glycolytic pathway to glycerol 3-phosphate
which is important for lipid biosynthesis. GPDH has previously been termed alpha glycerol-3-
phosphate dehydrogenase (alpha GPDH) and glycerol phosphate dehydrogenase. GPDH is also
referred to as DHAP reductase in algae and higher plants by reacting specifically with
nicotinamide adenine dinucleotide (NADH) and DHAP as substrates. GPDH functions as the
main link between carbohydrate metabolism and lipid metabolism and also make prominent
contributions to the electron transport chain in the mitochondria.
Diagram 1: Interaction between Carbohydrate and Lipid Metabolism
We can see that there is a link between carbohydrate metabolism (e.g Glycolysis) and lipid
metabolism. The intermediate that links these metabolic pathways is G3P and this is where the
enzyme Glycerol-3-phosphate dehydrogenase plays a significant role in the interaction. As
previously stated, Glycerol-3-phosphate dehydrogenase (GPDH) is the enzyme reversibly
catalyzing the reduction of dihydroxyacetone phosphate (DHAP) from the glycolytic pathway to
glycerol 3-phosphate hence why GDPH is so crucial for the interaction between the pathways.

8. From the absorbance values obtained from tubes 1,3 and 5 in particular estimate the
indigenous lactate and α-glycerol phosphate present in yeast.
So, for indigenous α-glycerol phosphate:

The concentration of α-glycerol phosphate is 0.5M since we used 1 mL the number of mols is
0.5 mol-----------------------1000mL
X ----------------------- 1mL
X = 0.5 mol * 1mL
1000 mL
= 0.0005 mol/mL

The concentration from tube 3 = 0.0005 mol/mL

Therefore, amount of indigenous α-glycerol phosphate = abs from 1* 0.0005mol/mL
abs from tube 3
0.383× 0.005 mol /mL
=
0.414
= 4.626×10-4 mol/ml

So, for indigenous lactate:

The concentration of lactate is 0.5M since we used 1 mL the number of mols is
0.5 mol-----------------------1000mL
X ----------------------- 1mL
X= 0.5 mol * 1mL
1000 mL
= 0.0005 mol/mL
The concentration from tube 5 = 0.0005 mol/mL

Therefore, amount of indigenous lactate = abs from 1* 0.0005mol/mL

abs from tube 5
0.383× 0.005 mol /mL
=
0.247
= 1.873×10-3 mol/ml
 Sources of error/limitation
 Colour intensity was judged based on vision, which may be inaccurate as two intensities
that may look the same are very different.
 Not fixing the solution out when reagents were initially added to the test tube. This could
have caused incorrect observation in colour intensity.
 There could have been contamination of micropipette tips when transferring the reagents
used in the experiment.
 Accidentally removing the pellet when removing the supernatant.
 Incorrect readings due to faulty spectrophotometer.

Conclusion
The activity of the dehydrogenase was evaluated by monitoring the colour change of methyl blue
from blue to colourless, as observed in test tubes B and C. The enzyme activity was measured by
observing how the intensity of the TTC colour shifted from colourless to red in all test tubes.
After that, the absorbance was measured, and test tubes 3 and 5 exhibited the highest absorbance
due to the addition of NAD to the solution.

References
BIOC 2014 Lab Manual (2021), Biochemistry Section, Department of Basic Medical Sciences,
The University of the West Indies, Mona Campus.

Farhana, A., & Lappin, S. L. (2022, May 8). Biochemistry, Lactate Dehydrogenase - StatPearls -
NCBI Bookshelf. Biochemistry, Lactate Dehydrogenase - StatPearls - NCBI
Bookshelf. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK557536/

Rustin, P., Munnich, A. & Rötig, A. Succinate dehydrogenase and human diseases: new insights
into a well-known enzyme. Eur J Hum Genet 10, 289–291 (2002). Retrieved from
https://doi.org/10.1038/sj.ejhg.5200793

Tanaka. (2021, April 1). Elucidation of the enzyme involved in 2,3,5-triphenyl tetrazolium
chloride (TTC) staining activity and the relationship between TTC staining activity and
fermentation profiles in Saccharomyces cerevisiae - PubMed. PubMed. Retrieved
October from https://pubmed.ncbi.nlm.nih.gov/33386278/