The document summarizes an experiment that investigated how altering temperature, adding inhibitors, and including cofactors impacts the rate of succinate dehydrogenase (SDH) enzyme activity. SDH plays a key role in the citric acid cycle by catalyzing the oxidation of succinate to fumarate. The experiment found that: - Changing the temperature away from the optimum decreased enzyme activity, likely due to lack of kinetic energy or denaturation. - Adding an inhibitor like sodium malonate decreased activity by slowing the reaction. - Including a cofactor like sodium succinate increased activity nearly 500% because cofactors act as catalysts.

1. Introduction
Succinate Dehydrogenase (SDH) is an enzyme
involved in the TCA cycle, a metabolic pathway
that constitutes the Complex II of the electron
transport chain, which function begins with
glycolysis and ends in the production of ATP
for aerobic respiration in the mitochondria.
(Rutter et al., 2010)
All enzyme activity
Figure 2 Comparison of the lowest and highest absorbance
obtained.
Figure 2 shows the comparison of tube A,
containing only Tetrazolium Red, phosphate buffer
and liver homogenate, and tube F which contains
all the components with no inhibitors. Tube A
shows a minimum absorbance of 0.031, whereas
tube F has an absorbance of 0.399.
Results
Figure 3 Difference in absorbance of 3 tubes (shown as bars)
containing an even concentration of components but incubated
at different temperatures.
As observed in figure 3, incubating the tubes at
different temperatures had a significant effect on
the absorbance at 440 nm. The results show that
37° C was the optimum temperature as it shows a
higher absorbance than the tubes incubated at
19° C and 65° C.
Adding an inhibitor
Figure 4 Difference in absorbance of 2 tubes containing the
same concentration of sodium succinate, but one of them (right)
contains sodium malonate
The addition of sodium malonate resulted in a
significantly lower absorbance at 400 nm as it can
be appreciated in figure 4. The absence of an
Department of Biosciences
Authors: Roger Coca Rocha
Department of Biosciences and Chemistry, Faculty of Health and Wellbeing,
Sheffield Hallam University, Sheffield S1 1WB, United Kingdom
Rate of Succinate Dehydrogenase activity under the
alteration of temperature and selective inhibition.
Materials and
Methods
1g of liver sample was homogenized with the
aid of ice-cold homogenization buffer and
the polytron homogenizer.
Specific concentrations of inhibitor (sodium
malonate), cofactor sodium succinate and
coenzyme NAD+ were added to 8 different
tubes to investigate the effects of the
presence or absence of these components in
the enzyme activity rate. All the tubes
contained an equal concentration of
Tetrazolium Red. A pH buffer was used where
necessary to add up to the final volume (2.5
ml).
To observe the effect of temperature change,
6 tubes were placed in a water bath at 37 C,
1 at room temp, and 1 at 65 C. All the tubes
were left incubating for 5 minutes and then 1
mL of liver homogenate was added to all the
tubes for a further 20 minutes incubation at
their corresponding temperature. All the
tubes were then placed on ice.
Discussion
Since absorbance is directly proportional to the rate of
enzyme activity, the following conclusions are made:
• Temperature: The rate of enzyme activity decreases
when the optimum temperature is altered. Either
denaturation or lack of kinetic energy.
• Inhibitors: The rate of enzyme activity decreases
with the presence of inhibitors because they bind to
SDH and slow down the formation of ESC.
• Cofactor: The addition of a cofactor results in an
increased rate of activity. This is because cofactors
bind to the enzymes and act as catalysts.
The highest increase in absorbance was shown when all
the components were added. The presence of coenzyme
NAD+ activates all other dehydrogenase enzymes
involved in. As a final conclusion, it is vital for
dehydrogenase enzymes in the liver to maintain an
optimum temperature and lack of inhibition.
References
• Daniel, R. M., & Danson, M. J. (2013).
Temperature and the catalytic activity of
enzymes: A fresh understanding. FEBS
Letters, 587(17), 2738–2743.
• Rutter, J., Winge, D. R., & Schiffman, J. D.
(2010). Succinate dehydrogenase –
Assembly, regulation and role in human
disease. Mitochondrion, 10(4), 393–401.
• Sharma, R. (2012). Enzyme Inhibition and
Bioapplications. IntechOpen.
• Zhang, C., Freddolino, P. L., & Zhang, Y.
(2017). COFACTOR: improved protein
function prediction by combining structure,
sequence and protein–protein interaction
information. Nucleic Acids Research, 45(W1),
W291–W299.
As illustrated in
figure 1, the
function of SDH is
to catalyse the
oxidation of
succinate into
fumarate by
reducing
coenzyme FAD
into FADH2.
Tetrazolium Red ‘steals’ the electrons from the
E.T.C. (granted by FADH2) and becomes reduced
itself resulting in a change to red. The intensity
of the colour allows for the quantification of
enzyme activity, which is proportional to the
absorbance. Different parameters can then be
changed to observe SDH behaviour.
Changing enzyme’s optimum temperature
should decrease its rate of activity due to lack
of kinetic energy or denaturation (Daniel &
Danson, 2013). Adding an inhibitor also
decreases the activity as it slows down the
formation on ESC (Sharma 2012). Finally,
adding a cofactor which acts as a catalyst
should increase the rate of activity (Zhang et
al., 2017).
0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
37.000 19.000 65.000
Absorbance
at
440
nm
Temperature °C
SDH activity under different temperature
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
No inhibitor Inhibitor
Absorbance
at
400
nm
SDH activity under the presence of an inhibitor
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
No cofactor Cofactor
Absorbance
at
440
nm
Enzyme activity in the presence of a cofactor
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Tube A Tube F
Absorbance
at
400nm
Effect of adding all the components
Acetone was added to each tube to stop the reaction.
The samples were centrifuged and the colour
changed was measured on a spectrophotometer at
440nm. The results were plotted in bar charts and
compared evenly
Figure 1. The role of SDH in the TCA
cycle and ETC
Effect of adding a cofactor
Figure 5 Difference in absorbance of 2 tubes (shown as bars)
one of which contains a cofactor (right) and the other doesn’t.
Figure 5 shows that when the cofactor sodium
succinate was added to the tube, this resulted
in an increased absorbance by 494% from 0.057
to 0.339.
Varying temperature