Succinate Dehydrogenase Activity of Mitochondria

Introduction:
The mitochondrion is often referred to as the powerhouse of the cell. It contains all of the
machinery needed to provide the cell and its components with energy to carry out cellular
processes. The matrix of the mitochondrion is where the TCA cycle occurs, in which pyruvate
(oxidized from glucose in gycolysis) is converted into acetyl-CoA, then fed into the pathway to
be oxidized to CO2 and its energy conserved. Succinate dehydrogenase is the only enzyme of the
TCA cycle that is also part of the electron transport system, thus, it is located in the inner
membrane. Succinate dehydrogenase and its conenzyme flavin adenine dinucleotiede (FAD),
represented as the complex E-FAD, oxidize the metabolite succinate to fumarate. Succinate
dehydrogenase removes electrons from succinate, which reduces FAD, thus reducing the enzyme
complex to E-FADH2. The reduced coenzyme then transfers the electrons to coenzyme Q to be
taken through the rest of the electron transport chain.
The purpose of this lab is to measure the rate of activity of succinate dehydrogenase
catalyzing the reaction succinate to fumarate in vitro using a mitochondrial fraction from
cauliflower cells.
The reaction is measured by observing the reduction of 2,6dinitrophenolindophenol (DCIP), an artificial electron acceptor, rather than coenzyme Q.
Adding sodium azide blocks the electron transport system so electrons cannot reduce coenzyme
Q. Instead, the electrons are transported from E-FADH2 to DCIP. The reduction of DCIP can be
identified by a color change; the oxidized form of the electron acceptor is blue and its reduced
form is colorless:
E-FADH2 + DCIPox (blue) E-FAD + DCIPre (colorless)
The extent to which DCIP is oxidized is measured by recording the different absorbances of
different enzyme concentrations using a spectrophotometer at 600 nm.
Tubes 1 through 3 are used to test the initial velocities of the reaction from succinate to
fumarate of different enzyme concentrations with the same amount of succinate. As enzyme
concentration changes, initial velocity should change also. Tube 4 is used to test the effect of a
competitive inhibitor (Malonate) on the activity of the enzyme. Tube 5 contains no added
sodium azide to block the normal path of electrons in the electron transport chain, and is
therefore used to compare reaction rates with those that contain sodium azide. Tube 6 is used to
test the reaction rate with no added succinate. Tube 7 is heated in a hot water bath to denature
succinate dehydrogenase and bring it to its inactivated state. It is then used as the 0-minute
reading mark for all the tubes to measure change in absorbance because as it cools, it transforms
into its native conformation, which is representative of the enzyme before the reaction occurs.
The changes in absorbance across 35 minutes should display the differences in reaction
rates based on differences in enzyme concentration. It should also show that the presence of
competitive inhibitors and absence of substrate and enzyme blockers (sodium azide) should slow
the rate of the reaction.

Materials and Methods:

Isolate mitochondria grind 20g cauliflower in chilled mortar with 40 mL cold mannitol
grinding medium and 5 g cold purified sand for 4 minutes.

Filter suspension with cheesecloth, centrifuge at 600 g at 4 C for 10 minutes.

Centrifuge postnuclear supernatant fluid at 10,000 g at 4 C for 30 minutes.

Discard postmitochondrial supernatant fluid, add 7.0 mL cold mannitol assay medium to
mitochondrial pellet.

Scrape pellet from tube, resuspend sediment in assay medium with pipet (make sure clumps are
completely dispersed). Keep in ice bath.

Label 10 cuvettes based on the following table from page 135 of lab manual. Heat 0.6 mL of
suspension for tube 7 in boiling water for 5 min, then cool in ice bath. Set spectrophotometer at
600 nm.

Add correct volume of assay medium and various solutions indicated from the table, but dont
add mitochondrial suspension. Cover tube with parafilm and invert to mix contents thoroughly.

Add correct volume of mitochondrial suspension to first cuvette (based on the following table),
then invert to mix thoroughly. Every 30 seconds, add mitochondrial suspension to the next
cuvette. Do not add mitochondrial suspension to blanks.

After 5 minutes, adjust spectrophotometer with blank #1 and take absorbance of tube 1. Adjust
again with blank #2 and take absorbance of tube 2, then again with blank #3 for tubes 3 7.

Take the absorbances for the seven tubes every 5 minutes for 35 minutes, always adjusting the
spectrophotometer using the blanks for their corresponding tubes.
Tube
Blank 1
1
Blank 2
2
Blank 3
3
4
5
6
7

Assay
Medium
3.7 mL
3.2 mL
3.1 mL
2.6 mL
3.4 mL
2.9 mL
2.7 mL
3.4 mL
3.4 mL
2.9 mL

Azide
(0.04M)
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
-0.5 mL
0.5 mL

DCIP
(5 x 10-4 M)

-0.5 mL
-0.5 mL
-0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL

Malonate
(0.2M)
------0.2 mL
----

Succinate
(0.2 M)
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
-0.5 mL

Mitochondria
l Suspension
0.3 mL
0.3 mL
0.9 mL
0.9 mL
0.6 mL
0.6 mL
0.6 mL
0.6 mL
0.6 mL
0.6 mL

Results:
Table 1: Absorbance Readings (A600) at Each Time Interval
Tube #
5 min
10 min
15 min
20 min
1
0.62
0.46
0.30
0.38
2
0.41
0.25
0.11
0.04
3
0.48
0.36
0.25
0.15
4
0.58
0.55
0.56
0.55
5
0.60
0.54
0.52
0.49
6
0.67
0.75
0.67
0.67
7
0.75
0.79
0.72
0.72

25 min
0.25
0.03
0.07
0.54
0.47
0.83
0.73

30 min
0.26
0.02
0.05
0.55
0.47
0.68
0.71

35 min
0.22
0.02
0.04
0.52
0.44
0.70
0.73

Table 2: Total Change in Absorbance (A) at Each Time Interval

Tube #
5 min
10 min
15 min
20 min
25 min
1
0.12
0.28
0.44
0.36
0.49
2
0.33
0.49
0.63
0.70
0.71
3
0.26
0.38
0.49
0.59
0.67
4
0.16
0.19
0.18
0.19
0.20
5
0.14
0.20
0.22
0.27
0.27
6
0.07
-0.01
0.07
0.07
-0.09
7
0.00
-0.05
0.02
0.02
0.01

30 min
0.48
0.72
0.69
0.19
0.27
0.06
0.03

35 min
0.52
0.72
0.70
0.22
0.30
0.04
0.01

Figure 1: Total Change in Absorbance at Each Time Interval for Tubes 1-4

Total Change in Absorbance at Each Time Interval

0.8

Absorbance

0.7

Tube 1

0.6

Linear (Tube 1)

0.5

Tube 2

0.4

Linear (Tube 2)
Tube 3

0.3

Linear (Tube 3)

0.2

Tube 4

0.1

Linear (Tube 4)

0
0

10

15

20

25

30

35

40

Time (minutes)

Figure 2: Initial Velocity of Enzyme Concentrations for Tubes 1-3

Initial Velocity of Enzyme Concentrations

0.07

0.07

0.06
0.05

0.05
0.04
Initial Velocity (A/min)

0.03
0.02

0.02

0.01
0
0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Enzyme Concentration

Table 1 displays the absorbance readings at each 5-minute time interval for 35 minutes.
Tubes 1 through 5 have an almost identical pattern of starting with a high absorbance reading
and decreasing at each time interval. Tubes 6 and 7 have an irregular pattern and do not
consistently increase or decrease throughout the time frame. Table 2 displays the changes in
absorbances at every 5-minute interval. The data for every time interval of every tube was
collected by subtracting the reading of each tube at the specified time from the absorbance

reading from the 5-minute interval for tube 7. Tubes 1, 2, 3, and 5 have a general pattern of
starting with a lower difference at 5 minutes, then the changes increase, and then eventually level
off and plateau. Tube 4 tends to fluctuate between 0.19 and 0.22. Tubes 6 and 7 do not have a
consistent pattern of change.
Figure 1 displays the changes in absorbances for every 5-minute interval for tubes 1
through 4. In accordance with Figure 2, it shows that tubes 1 through 3 tend to increase for a
while, and then level off, and tube 4 stays pretty constant for the entire 35 minutes. Figure 2
shows the initial velocities of tubes 1, 2, and 3. It exhibits that as enzyme concentration
increases in each tube, initial velocity increases as well.
Discussion:
According to the Michaelis-Menton equation, initial velocity is directly proportional to
enzyme concentration. With more of the enzyme present, more substrate will bind to that
enzyme quicker because of the enzymes high affinity for the substrate, thus having a higher
initial velocity of the reaction. This can be seen in the data from Figure 2, which shows that as
enzyme concentration increases, the initial velocity of the reaction from succinate to fumarate via
succinate dehydrogenase increases as well. Tube 1 has the least amount of mitochondrial
suspension, which as the least amount of succinate dehydrogenase, thus it has the lowest initial
velocity; tube 2 has the highest initial velocity because it has the highest enzyme concentration in
0.9 mL of mitochondrial suspension.
Malonate, a competitive inhibitor, was added to tube 4 to test its effect succinate
dehydrogenase and the reaction of succinate to fumarate. Tube 3 and tube 4 have the same
enzyme concentration, but tube for has a slower reaction rate because of malonates interference
with the enzyme. The competitive inhibitor has a similar molecular structure to succinate, so it
can bind to the active site of succinate dehydrogenase, preventing succinate from binding. This
creates a slower initial velocity and reaction rate because it takes longer for succinate to bind to
its enzyme.
The changes in absorbance in table 2 describe the rate of the reaction from succinate to
fumarate via succinate dehydrogenase. The increasing changes indicate that more succinate
keeps being oxidized to fumarate, and E-FAD is being reduced to E-FADH 2. The change in
absorbances along with the subsequent color changes in the tubes from blue to colorless indicates
that E-FADH2 is oxidized to reduce DCIP. Toward the end of 35 minutes, the changes start to
level off and plateau, indicating that less succinate is being bound and converted because it is all
being converted, as seen in figure 1. However, in tube 6, there was no succinate added, which
lead to a slower reaction rate because there was less succinate present in the mitochondria
compared to the other samples.
In tube 5, sodium azide was not added to block the electron transport system to monitor
the reduction of DCIP. Reduction of DCIP was noticed by a change in absorbance. The changes
in tube 5 are significantly less compared to those with sodium azide because more electrons from
E-FADH2 are being transferred to coenzyme Q rather than DCIP.
The mitochondrial suspension for tube 7 was heated for 5 minutes in order to denature
succinate dehydrogenase and bring it to its inactivated state. As it cooled, it transformed to its
native conformation, which was representative of the enzyme right before the reaction from
succinate to fumarate. Its 5-minute absorbance reading was used as the 0-minute reading to

determine change in absorbance in all the tubes because it is present in its native conformation
without having gone through the reaction.
Based on all the data that has been collected, it is easy to conclude that as the
concentration of succinate dehydrogenase increases, the rate that succinate is oxidized to
fumarate increases as well. However, addition of malonate and reducing the amount of succinate
or sodium azide slows down the reaction rate because of their interactions with succinate
dehydrogenase.
Bibliography:
Bregman, Allyn A. Laboratory Investigations in Cell and Molecular Biology. John Wiley &
Sons, Inc., New York, 2002.
Karp, Gerald. Cell and Molecular Biology: Concepts and Experiments. John Wiley & Sons,
Inc., New Jersey, 2008.

Succinate Dehydrogenase
Activity of Mitochondria

Cassandra Saikin
Cell Biology Lab
Dr. Barbera
March 18, 2008