The document summarizes an experiment that investigated the effects of temperature, inhibition, and coenzyme addition on the enzymatic activity of succinate dehydrogenase (SDH). It found that increasing substrate concentration counteracted the inhibitory effects of malonate. Adding the coenzyme NAD+ increased SDH activity by approximately fourfold. Activity was highest near the optimal temperature of 80°C for SDH and much lower at room temperature due to enzyme denaturation at suboptimal temperatures.

1. Results and
Discussion
The effect of increased substrate
concentration against inhibition
Tubes C and D shown below in Figure 1
showed the effect of increased substrate
concentration (Tube D – Orange) on the
activity of SDH, when the inhibitor malonate is
present. Tube B acts as a control.
Department of Biosciences
Authors: Rebecca Lindley
Department of Biosciences and Chemistry, Faculty of Health and Wellbeing,
Sheffield Hallam University, Sheffield S1 1WB, United Kingdom
Investigating the effect of temperature, inhibition and coenzyme
addition on enzymatic activity of oxidative metabolism in a liver
homogenate
Materials and
Methods
An enzyme assay was carried out where
liver samples were minced and
homogenised to remove cellular
contents.
Different volumes of sodium succinate,
sodium malonate, NAD+ and phosphate
buffer were added to equal amounts of
tetrazolium red and liver homogenate in
each tube.
Tubes A-F were placed in a water bath
at 37°C, Tube G left at room
temperature and tube H kept at 65°C.
pH was stabilised via the addition of
phosphate buffer to each sample.
Acetone was added to each sample to
stop the reaction . The samples were
then centrifuged at 2,500 rpm at 5
minutes. A spectrophotometer was set
at 440nm and blanked using water in a
glass cuvette. Absorbance of each
sample was measured and recorded.
Conclusions
In conclusion, the higher the temperature the
enzyme is working at, the faster the rate of
enzymatic activity. The addition of substrate will
counteract the effect of malonate as an inhibitor
and increase activity of SDH by approximately
tenfold. The addition of a coenzyme will increase
enzyme activity by approximately fourfold.
Therefore, it is observed that the enzyme SDH
would work fastest and most efficiently at 80°C,
with NAD+ added and with increased substrate.
This experiment therefore determined the effects
of different variables on enzymatic activity and
completed its aim.
References
1. Fernandes, A. S., Pereira, M. M., & Teixeira, M. (2001). The
succinate dehydrogenase from the thermohalophilic bacterium
Rhodothermus marinus: redox-Bohr effect on heme
bL. Journal of bioenergetics and biomembranes, 33(4), 343–
352. https://doi.org/10.1023/a:1010663424846
2. Britannica, T. Editors of Encyclopaedia (2013, May
16). Tricarboxylic acid cycle. Encyclopedia Britannica.
https://www.britannica.com/science/tricarboxylic-acid-cycle
3. Green, J., & Narahara, H. (1980). Assay of succinate
dehydrogenase activity by the tetrazolium method: evaluation
of an improved technique in skeletal muscle fractions. Journal
Of Histochemistry & Cytochemistry, 28(5), 408-412. doi:
10.1177/28.5.696664
Introduction
This aim of this experiment was to investigate
the effects of temperature, coenzyme and
inhibition on the enzyme SDH (Succinate
Dehydrogenase). The hypothesis is that as
temperature increases activity increases until
80°C1 (A S Fernandes, M M Pereira, M Teixera, 2001, which
has been found to be the optimum temperature
for SDH. Addition of malonate should reduce
activity and therefore absorption, and addition
of coenzyme should increase activity.
The image below shows the stages of the TCA
cycle, where SDH catalyses the oxidation of
succinate into fumarate. The TCA cycle occurs
in the matrix of the mitochondria, therefore
liver homogenate was used in this experiment
as the liver has a high density of mitochondria
due to its more frequent contraction than other
organs. Tetrazolium red therefore can be used
to measure rate of enzymatic activity.
Enzymes must work
efficiently for a fast
rate of hydrolysis of
substrate, which is
only possible in
optimum conditions
to avoid
denaturation.
2 (Britannica, T. Editors of Encyclopaedia (2013, May 16))
Figure 1 – An enzyme assay was carried out to
investigate effect of competitive inhibition on activity of
the enzyme SDH, and how increasing substrate
concentration affected inhibition. Standard deviation is
shown in error bars.
Succinate concentration was increased twofold
between tubes C (blue) and D (orange). In
Figure 1, it can be seen that mean absorption
of SDH increases by 10.67x upon doubling
substrate concentration. This supports the
hypothesis made that malonate reduces
activity but is counteracted by increasing
substrate concentration.
Effect of addition of coenzyme
Tubes E and F, in comparison with tubes A
and B, shown below in Figure 2, show that
addition of coenzyme NAD+ increases
activity of the SDH enzyme.
Figure 2 – An enzyme assay was carried out to
investigate the effect of adding the coenzyme NAD+
to the samples. A and E, and B and F can be
compared as they are most otherwise similar
respectively. Standard deviation is shown in error
bars.
E had no substrate so addition of coenzyme
made no difference, however in tube F there
was substrate and addition of NAD+
increased enzyme activity 4.33x. This
supports the hypothesis that increased
concentration of NAD+ increases enzyme
activity. Figure 2 above therefore shows that
SDH activity increases as NAD+
concentration increases.
The effect of temperature on SDH activity
Tubes G and H shown below in Figure 3 highlight
the effect of temperature on enzymatic activity,
low temperature (19°C in tube G) and high
temperature (65°C in tube H). Test tube B acts
as a control.
Figure 3 – An enzyme assay was carried out to
investigate effect of temperature on SDH activity.
Optimum temperature for SDH is 80°C. 1 (A S Fernandes,
M M Pereira, M Teixera, 2001) Standard deviation is shown in
error bars.
As tube H was closer to optimum temperature for
SDH, Figure 3 shows that the closer to optimum
temperature, the faster enzymatic activity as
there is less/ no denaturation of enzymes. At
19°C most, if not all, enzymes are denatured
and therefore non-functional. Figure 3 therefore
supports the hypothesis as it proves that
optimum temperatures increase enzymatic
activity.
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