Succinate Dehydrogenase Assay: Measurement of Enzyme Activity

SUCCINATE DEHYDROGENASE ASSAY
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SUCCINATE DEHYDROGENASE ASSAY
Introduction
The mitochondrion is usually known to be the powerhouse of any living cells in the body of an
organism. It is composed of all the required machinery in the provision of the cells as well as
their components with the energy that is needed to carry out the various cellular processes
including movement, growth and development among other vital processes of the cell. The
matrix of the mitochondrion forms the base on which TCA cycles takes place, where pyruvate
which is oxidize from glucose in glycolysis processes ins changed into acetyl-CoA, which is then
fed into the pathway to undergo oxidation releasing carbon dioxide and the conserved (Aspuria
et al., 2014).
Succinate dehydrogenase remains the only enzyme rom the TCA cycle which is as well part of
the electron transport system hence it is often located on the inner membrane. Succinate
dehydrogenase has a co-enzyme, flavin adenine dinucleotiede all of which are represented as a
complex E-FAD and work by oxidizing the metabolite succinate to form fumarate. Succinate
dehydrogenase eliminates the electrons from succinate leading to a reduction in FAD and thus a
decrease in the enzyme complex E-FADH2. The reduced enzyme in turn transfers electrons to
coenzyme Q from which it is takes through the remaining chain of electron transport (Calió et
al., 2017).
Succinate dehydrogenase is an enzyme that is located in the inner membrane. The location
makes it a very easy target for isolation when conducting a study on the citric acid cycle. The
main role of the enzyme in the cell is to oxidize succinate to form fumarate which is then usable
as a marker during the process of isolation of mitochondria via differential centrifugation. The

isolated mitochondria can then be treated using a sodium azide reagent to prevent transportation
of electron of the mitochondrion in the cell extract.
An artificial electron acceptor is often used in taking the measurement of the activity of an
enzyme (2, 6-dichlorophenolindphenol, DCPIP), which is used in the acceptance of two
electrons. Upon reception of electrons, the oxidised 2, 6-dichlorophenolindphenol undergoes
reduction and the colour of the mixture turns to colourless from blue (Dudek et al., 2015). The
change in the colour can be quantified suing spectrometry at a range of 600nm which then
provides the contents of mitochondria in the given sample.
Enzymes serve as regulators for the various metabolic pathways which reduce the activation
energy so as to catalyze acceleration in the rate of biochemical reactions. Most of the enzymes
are characterized as demonstrating Michaelis-Menten (M-M) kinetic characteristics. Enzymes
work by creating a binding with their substrates in a reversible manner thereby altering the
conformation of the substrate leading to the formation of a complex of enzyme-substrate and
hence detaching resulting in free enzyme and the product. In cases where the concentration of the
substrate is low, little enzyme activity is noticed and thus a slow rate of reaction (Guzzo et al.,
2014).
On the contrary, a high concentration of the substrate or when the substrate has a saturated
concentration, the enzymes tend to be more active and hence a faster rate of reaction. At some
point when the substrate concentration is highly saturated, where is recorded no increase in the
rate of the reaction. Alongside the concentration of the substrate, these changes could be featured
as the Michaelis-Menten (M-M) constant as well as the maximum velocity (Lampropoulou et al.,
2016). These factors form an integral part of the factors which influence the initial velocity of

any biochemical reactions and thus contributing to an elaborate understanding of the Michaelis-
Menten (M-M) equation.
Nonetheless, in the presence of a competitive inhibitor, the inhibitor is able to bind to the active
site as to stop the normal substrate from making a bind and forming the product. This leads to a
competition between the substrate and the product for the active site of the enzyme, a dynamic
that is based on the Michaelis-Menten (M-M) equation and permits the maximum velocity to
remain constant even as the Michaelis-Menten (M-M) constant changes (Kim et al., 2015).
The role of this laboratory experiment was measurement of the activity rate of succinate
dehydrogenase in the catalysis of the reaction succinate to form fumarate in vitiro with the aid of
mitochondrial fraction extracted from cauliflower cells. The measurement of the rate of the
reaction is done through making observations on the reduction of 2, 6-dichlorophenolindphenol
which is an artificial electron acceptor as opposed to coenzyme Q.
Sodium azide blocks are added to the electron systems to ensure that the electrons are
empowered to reduce the concentration of coenzyme Q (Kitazawa et al., 2017). The electrons are
rather transported to 2, 6-dichlorophenolindphenol from E-FADH2. A colour change of the 2, 6-
dichlorophenolindphenol would be used to identify its reduction where the oxidized form of the
acceptor is often blue in colour which changes to colourless upon being reduced. The equation of
the reduction is as shown below:
E-FADH2+DCPIPox (blue) E-FADH2+DCPIPre (colourless)
The degree of oxidation of the 2, 6-dichlorophenolindphenol DCIP is measured using the various
absorbances of the various concentrations of enzymes through the use of a spectrophotometer at
600nm.