Oral Presentation

1. Department of Biosciences
Introduction
Continuous production of energy is a vital process
in all living organisms. The most common energy
form, ATP (adenosine triphosphate) is synthesized
in a series of stages through the aerobic cellular
respiration process, one of which is the Krebs
Cycle, also known as the Citric Acid Cycle (Hardin,
Bertoni & Kleinsmith, 2017).
The Krebs cycle is a biochemical pathway that
aids ATP synthesis through oxidative metabolic
reactions catalyzed by a series of enzymes and
coenzymes (Kalyani & Chanchal, 2013) (Figure 1).
It has been shown that enzymatic reactions occur
under various conditions due to enzymes being
specific catalysts that are affected by pH and
temperature and are sensitive to inhibitors
(Madhumathi et al., 2007).
The aim of this experiment was to analyze the
impact of temperature variation and an inhibitor,
which were expected to greatly influence SDH
(succinate dehydrogenase) activity, which is one
dehydrogenase enzyme used in the Krebs Cycle.
Moreover, the effect of NAD+ (nicotinamide
adenine dinucleotide) coenzyme was analyzed,
taking into consideration that all experiments
were conducted on a liver homogenate sample
that lacked the presence of this coenzyme.
Materials and Methods
Preparation of liver homogenate
The liver sample was minced and mixed with
ice-cold homogenization buffer, then the
sample was homogenized.
Tubes containing different components
were prepared
Eight tubes labeled from A to H were
prepared and tetrazolium red was added in
each of them. Tetrazolium red and the
phosphate buffer (pH 7.2) was added to
tube A. Sodium succinate was added to B-H
tubes, except E. Sodium malonate was only
added in tubes C and D. NAD+ was added in
tubes E and F.
Incubation time
The tubes were left to incubate for 5
minutes at different temperatures as
follows:
• tubes A-F at 37°C
• tube G at 19°C
• tube H at 65°C
Adding the liver homogenate sample
Following this step, the liver homogenate
sample was added to each tube and left to
incubate again for another 20 minutes.
Acetone was added to each tube, which
were then centrifuged for 5 minutes at
2,500 rpm. The absorbance was then
quantified using a spectrophotometer at
440 nm.
Results and Discussion
Temperature variations
As shown in Figure 2, samples B, G and H
were incubated at different temperatures
for 5 minutes. The highest absorbance value
was identified for sample B (37°C) and the
lowest for sample G (19°C).
Malonate inhibition
Figure 3 shows a very high absorbance
value for sample B which lacked malonate,
compared to low values for samples C and
D which contained malonate in the same
concentration, but succinate in higher
Authors: Adina Georgiana Dorobantu
Department of Biosciences and Chemistry, Faculty of Health and Wellbeing,
Sheffield Hallam University, Sheffield S1 1WB, United Kingdom
The effect of temperature variation and the presence of the
malonate inhibitor and NAD+ coenzyme on SDH activity
concentration for sample D.
Presence of NAD+
In Figure 4, the presence of NAD+ and sodium
succinate in sample F, along with the presence
of only NAD+ in sample E was observed in
comparison to sample B, which only contained
sodium succinate.
Conclusions
Enzyme denaturation (Figure 2) - SDH activity
was inactive at 19°C (sample G) and temporarily
active at 65°C (sample H) until reaching
denaturation (Purich, 2010) (Madhumathi et al.,
2007). The optimum SDH activity was registered
at 37°C.
Malonate inhibition – Competitive inhibition was
observed for sample C due to inactive SDH
activity, while non-competitive inhibition was
present in sample D, which only slowed down
SDH activity (Purich, 2010) (Figure 3).
Effect of NAD+ - SDH activity was relatively low in
the succinate sample and very low in the NAD+
sample. However, a very high value was observed
in sample F, due to the presence of both
succinate and NAD+, which reflected the total
dehydrogenase activity (Purich, 2010) in the liver
homogenate (Figure 4).
The human body creates the optimal conditions
for enzymatic activity, a complex process
enhanced by this experiment.
Another interesting aspect that could be taken
into consideration in future work on this subject,
would be to analyze the reaction time of enzymes
under different environmental conditions such as,
pH, temperature or activators and inhibitors.
References
Hardin Jeff, Bertoni Paul Gregory & Kleinsmith J. Lewis (2017)
Becker’s World of the cell, Global Edition, 9th ed., Pearson
Education, Limited, pp. 274-275
Kalyani Korla & Chanchal K. Mitra (2013) “Modelling the Krebs
cycle and oxidative phosphorylation”, Journal of Biomolecular
Structure and Dynamics; https://www-tandfonline-
com.hallam.idm.oclc.org/doi/full/10.1080/07391102.2012.762723
Madhumathi M., Cheerla Srinija, Saravanabhavan S.,
Thanikaivelan P., Rao Raghava J., Chandra Babu N. K. &
Balachandran Unni Nair (2007) Factors Influencing Activity of
Enzymes and Their Kinetics, Bioprocessing of Skin, Humana Press
Inc.
Purich L. Daniel (2010) Enzyme Kinetics: Catalysis & Control, A
Reference of Theory and Best-Practice Methods, Chapter 7, pp.
379-484
Figure 1. Metabolic reactions occurring during the
Krebs Cycle (Hardin, Bertoni & Kleinsmith, 2017).
Complex enzymatic steps lead to the formation of ATP. 37°C
19°C
65°C
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
B G H
Figure 2. SDH activity at different temperatures.
Sample B was maintained at room temperature,
sample G at 19°C and sample H at 65° C.
-0.020
-0.010
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
B C D
Figure 3. The effect of the inhibitor malonate.
Malonate was only added to samples C and D.
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
B E F
Figure 4. The effect of NAD+ presence and the overall
SDH activity. Sodium succinate was added to samples B
and F, while NAD+ was only added in samples E and F.