Title: Examination of succinic acid dehydrogenation

Introduction
The mitochondrion is often referred to as the powerhouse of the cell because it
contains all of the machinery needed to provide the cell and its components with
energy to carry out cellular processes. In the matrix of the mitochondrion this is
where the citric acid cycle also known as the tricarboxylic acid cycle or the
Krebs cycle occurs, in which pyruvate glycolysis is converted into acetyl-CoA,
then fed into the pathway to be oxidized to carbondioxide and its energy
conserved in this cycle one of the reactions converts succinate to forming
malate in which the reaction is catalysed by succinic dehydrogenase with an
FAD+ covalently attached to it (Walker and Wilson, 1994).

To understand what happens next, it is important to remember that FAD+ is an

energy transfer molecule. It only lasts a very short time and must offload what it
has picked up as quickly as possible so that there are sufficient energy transfer
molecules available for the transfers to continue as more reactions are
catalysed.in this reaction the FAD+ is reduced when it accepts the two
hydrogens from the succinic acid, but it is immediately oxidized when it
transfers those two hydrogens to fumaric acid. Since citric acid cycle is an
enzymes catalysed metabolic pathway it is important understand how fast the
reactions would be when some new substances are introduced in the system and
this can be done by using inhibitors for example in the citric acid cycle malonic
acid can be used since it has the same structure as succinic acid,hence its effect
can be observed by various chemical methods ( Ashworth , et al.,1974).

Aim:

To examine the metabolic rate of yeast by observing the effect of each of the
two acids on the reaction rate in which it was determined by how fast methylene
indicator changes its colour from blue to colourless, the faster the blue colour
disappears the faster the rate.
Materials and methods
Nine test tubes were labelled with letters from A to I and the test tubes were
prepared with different solutions as shown by the table below.
Table 1: a tabular representation of reagents used in the determination of the
metabolic rate of yeast in which the concentration of both sugars and acids was
0.5M and also reflects the temperature at which incubation was carried out at
Tub
e#
A
B
C
D
E
F
G
H
I

Yeast
suspensio
n
1ml
1ml
1ml
1ml
1ml
1ml
1ml
1ml
1ml

Glucos
e

Fructos
e

1ml
1ml
1ml
1ml
-

1ml
1ml
1ml
1ml

Succini
c
acid
1ml
0.5ml
1ml
0.5ml
-

Maloni
c
Acid
1ml
0.5ml
1ml
0.5ml
-

Wate
r
2ml
1ml
1ml

Incubation
temperatur
e
o
37 C
37oC
37oC
37oC
37oC
37oC
37oC
37oC
37oC

Exactly 100l of 0.1% methylene blue was added to each of the test tubes and
the mixture was mixed on a vortex giving a light blue solution. Volume of 500l
mineral oil was added to each test tube while the test tubes were held at an angle
so that the oil can form a film layer over the top. This process was done so as to
slow the diffusion of oxygen into the system .all the test tubes were incubated at
37C and observations of colour changes were made and recorded at 5 minute
intervals

Results
Attached

Discussion
It is now well established that the oxidation of metabolites by living
cells involves the passage of hydrogen or electrons or both along a series of
oxidation-reduction systems. The oxidation of a substrate, thus, is accomplished
by the removal of hydrogen atoms which pass to a hydrogen acceptor. This
reaction proceeds only in the presence of enzymes which are usually specific for
the substrate and sometimes specific for the acceptor, such enzymes being
classed as dehydrogenases. One of the reactions in citric acid cycle involves
such type of oxidation reactions in which the hydrogen atoms are removed from
succinate to form fumarate and the enzyme that catalyse that reaction is
succinate dehydrogenase, where FAD that is covalently attached to this enzyme
is a hydrogen acceptor molecule. For this practical we used methylene blue in
place of FAD as an acceptor for hydrogen atoms the reason for that was for us
to be able to monitor the progress of the experiment in the sense that this
indicator is blue in colour before it is reduced but when it is reduced its colour
will change from blue to colourless thereby we can determine the metabolic rate
by observing the time taken for that blue colour to disappear ( Dennis, et
al.,1995).

The aim of this practical was to examine the effect of malonic acid and succinic
acid on the metabolic rate of the yeast, malonic acid was chosen because it has a
structure that is very similar to succinic acid therefore when we apply the lock
and key hypothesis proposed by Emil Fischer we can say that this substrate will
also be capable of binding to succinate dehydrogenase hence it can also be
placed in the citric acid cycle along with succinic acid for us to observe its
effect in the cycle. From the results we got nothing changed in tube A, there was
a milky suspension and a blue layer which suggests the absence of succinic acid
for reducing methylene blue indicator. In tube B nothing changed and this
happened for the rest of the test tubes they gave the same result as in tube (
Alicia , et al.,2002).

Based on theory we were expecting the metabolic process to be high in tube B

because there was no malonic acid in that tube to act as an inhibitor, in tube C
and the rest of the tubes without succinic acid we were expecting the light blue
colour of an indicator to remain there because there because there was no source

of hydrogen to reduce methylene blue. In tube D and G the blue colour was
supposed to disappear as well but at slower rate due to inhibition caused by
malonic acid, however we couldnt be able to observe the metabolic rate in tube
D and G, the failure for the blue colour to disappear can be as a result of using
excess methylene blue such that the hydrogen atoms from succinic will be
insufficient to reduce it ( Banowetz , et al., 1996).

References
Alicia N, Norman R, Jones F, 2002, principles and techniques of
biochemistry, 4th edition, page 402-406.

Ashworth B, Brewer M , Pesce J , 1974 , experimental techniques in

biochemistry, page 38-39.

Banowetz S, Jodi K and Hancock L, 1996, biochemistry and molecular

biology, 5th edition, page 56.

Dennis V , Geoffrey Z , William P , 1995 , principles of biochemistry ,

page 83-85.
Walker J and Wilson K, 1994, principles and techniques of practical
biochemistry, 4th edition, page 18 -20.