The document describes an experiment that measured the activity of the enzyme succinate dehydrogenase (SDH) in liver tissue under different conditions. SDH activity was measured using a triphenyl tetrazolium chloride (TTC) assay and spectrophotometer. The results showed that SDH activity decreased when the competitive inhibitor malonate was added. SDH activity also decreased at temperatures lower and higher than the optimal 37°C. The addition of NAD+ increased overall enzymatic activity but did not specifically increase SDH activity. The aim was to test how SDH activity is affected by malonate, temperature, and NAD+.

1. TTC Assay
1 mL of liver homogenate was added to
each tube, then incubated again for a
further 20 minutes.
7 mL of acetone was added to each tube,
then centrifuged at 2500 rpm before being
measured at 440 nm on a
spectrophotometer.
Results and Discussion
The mean absorbency results for each
tube from Table 1, in the TTC Assay were
plotted into a bar chart in order to compare
activity. The data came from repeat
experiments and the mean, standard
deviation and the coefficient of variation
were calculated for each tube as shown in
Table 2.
Table 2. Table depicting the mean
absorbency values at 440 nm, the standard
deviation and the coefficient of variation for
each sample tube.
The Effect of Malonate on SDH Activity
The data shows that when Malonate is
added, SDH activity is inhibited as Figure
2 shows. The only difference between
tubes B and C is the addition of Malonate.
Both tubes were incubated at 37°C.
Figure 2. Bar chart showing tube B with no
inhibitor vs tube C which contained a
competitive inhibitor; Malonate. Tube B had
an absorbency of 0.116 at 440 nm whereas
tube C had an absorbency of 0.028.
Standard Deviation error bars have been
added, showing the variation in data
obtained. The standard deviation was
calculated by using Excel.
The coefficient of variation was calculated
for each tube by dividing the standard
deviation by the mean then multiplying by
100%, for example, for tube B, the mean
absorbency was 0.116 and the standard
deviation was 0.070.
0.070 / 0.116 x 100% = 60.345%.
Succinate Dehydrogenase; The Effects of
Malonate, Temperature and NAD+ on its Activity
Methods
Preparation of the Liver Homogenate
1g of Liver with 20 mL of homogenisation
buffer was homogenised and kept on ice.
Sample Preparation for TTC Assay
8 falcon tubes were prepared and
labelled A – H as shown in Table 1.
Table 1. Falcon tubes prepared showing
components and volumes of each in mL.
The tubes were incubated at different
temperatures ranging from 19 - 65°C.
References
Chhabra, N., 2021. Succinate to Fumarate. [image] Available at:
<https://www.ourbiochemistry.com/impaired-tca-cycle-enzyme-
activity-in-angular-stomatitis/>
Rustin, P., Munnich, A. and Rötig, A., 2002. Succinate
dehydrogenase and human diseases: new insights into a well-
known enzyme. European Journal of Human Genetics, 10(5),
pp.289-291.
Tymoczko, J., Berg, J. and Stryer, L., 2015. Biochemistry. 3rd
ed. New York: W.H. Freeman and Company, p.349.
Introduction
Succinate Dehydrogenase (SDH) is an
Enzyme in the Tricarboxylic Acid Cycle
which aerobic cells cycle through to
produce energy: ATP. SDH catalyses
oxidation of succinate into fumarate
using FAD as a coenzyme which gets
reduced, becoming FADH2. (Tymoczo et
al,, 2015). Figure 1 shows this reaction.
Figure 1. Oxidation of succinate into
fumarate showing the reduction of the
coenzyme FAD into FADH2. (Chhabra,
2021)
SDH activity can be measured by a
Triphenyl Tetrazolium Chloride (TTC)
Assay and quantified on a
spectrophotometer.
The aims of the experiment were to
measure the amount of SDH activity in
a liver homogenate, and how Malonate,
temperature and a different coenzyme,
NAD+ affects its activity. The hypothesis
is that Malonate will reduce the SDH
activity as will high or low temperatures
and that NAD+ will increase overall
enzymatic activity but not SDH
specifically.
This reflects a great deal of variation in the
experiment, and therefore shows the experiment
wasn’t conducted precisely.
The Effect of Temperature on SDH Activity
Tubes G and H were incubated at 19°C and
65°C respectively, and the absorbency results
show that SDH activity was reduced compared to
tube B which was incubated at 37°C as shown in
Figure 3.
Figure 3. Bar chart showing tube B incubated at 37°C
vs tube G at 19°C and tube H at 65°C. The chart
shows SDH activity decreases at lower and higher
temperature than 37°C. Standard deviation error bars
were added to show variability of the data as shown in
Table 2.
The Effect of NAD+ on SDH Activity
Tube F had NAD+ added and had the highest
absorbency. The experiment hasn’t shown much
qualitative data in terms of SDH activity only due to
other enzymatic reactions taking place in the liver
homogenate.
Department of Biosciences
Author: Alicia Gasston
Department of Biosciences and Chemistry, Faculty of Health and Wellbeing,
Sheffield Hallam University, Sheffield S1 1WB, United Kingdom
Conclusions
The aims of the experiment were to measure SDH
activity under different parameters. The results
clearly evidence the hypothesis that SDH activity is
inhibited by Malonate and lower/higher
temperatures than 37°C.
The addition of NAD+ doesn’t generate qualitative
results as NAD+ requires more energy to be
reduced by SDH therefore the absorbency
measurement doesn’t reflect SDH activity, but
rather several different enzymatic reactions.
The activity of SDH is important in research as it’s
linked to several diseases like paraganglioma due
to mutations of the SDH gene. Future work can be
conducted looking at why it causes a reduction in
SDH activity. (Rustin et al,, 2002).
A B C D E F G H
Tetrazolium red 0.1
mol/L
1 1 1 1 1 1 1 1
Sodium succinate 0.2
mol/L
--- 0.5 0.5 1 --- 0.5 0.5 0.5
Sodium malonate 0.1
mol/L
--- --- 0.5 0.5 --- --- --- ---
NAD
+
0.01 mol/L --- --- --- --- 0.5 0.5 --- ---
Phosphate buffer 0.1
mol/L, pH 7.2
1.5 1 0.5 --- 1 0.5 1 1
A B C D E F G H
Absorbency
@ 440 nm
0.031 0.116 0.028 0.063 0.057 0.399 0.032 0.074
Standard
Deviation
0.006 0.070 0.013 0.013 0.025 0.187 0.019 0.065
Coefficient of
Variation %
19.355 60.345 46.429 20.635 43.860 46.867 59.375 87.838