Lab 6 - CELLULAR RESPIRATION: THE CITRIC ACID CYCLE

REACTIONS
In chapter 6 (A Tour of the Cell) you learned that the function of mitochondria is to produce
energy in the form of ATP for cell use. The process that converts the energy in food molecules
into the energy of ATP is known as cellular respiration. Our current understanding of the
respiration process suggests that there are several independent but interconnected stages
involved. These include:
1.
2.

3.

4.

Glycolysis: food molecules (typically considered to be the sugar glucose) are converted
to three-carbon molecules called pyruvate.
An Intermediate Step: a complex reaction that essentially results in 1) the oxidation of
pyruvate to generate acetate, 2) the attachment of coenzyme A to acetate to produce the
molecule known as acetyl CoA, and 3) the release of CO2 and NADH (a reduced electron
carrier). Acetyl CoA is the starting point for the citric acid cycle.
The Citric Acid Cycle (or Krebs Cycle or TCA Cycle): a series of reactions in which the
acetyl CoA is broken down one step at a time to eventually produce carbon dioxide and
in the process generate reduced electron carriers (e.g. NADH and FADH2).
The Electron Transport System (ETS): a series of oxidation-reduction reactions that use
the transport of electrons to generate energy needed to phosphorylate ADP to produce
ATP.

Your text book (see Chapter 9) reports the location where each of the major stages of cellular
respiration occurs. Glycolysis occurs in the cytoplasm of cells, while the others occur in
mitochondria. Mitochondria are a double membrane-bound organelle. The interior membrane is
folded into repeated structures called cristae, and it is here that the ETS is located. The citric
acid cycle occurs largely within the mitochondrial matrix (the liquid region created/surrounded
by the inner membrane).
In this lab exercise, you will perform a study similar to that performed by scientists several years
ago to explore the cellular location of the citric acid cycle. A protocol involving differential
centrifugation is available for isolating mitochondria from plant tissues. Succinate
dehydrogenase is an enzyme used to catalyze a reaction in the citric acid cycle. The study that
you will perform will test the following hypothesis:
The citric acid cycle occurs within mitochondria.
Again, to test this hypothesis you will look for the presence of one of the enzymes that functions in
this cycle, namely succinate dehydrogenase (SDH).
The initial reaction catalyzed by succinate dehydrogenase is summarized as follows:
Succinate + FAD -----> fumarate + FADH2
In vivo, the reduced form of FAD (e.g. FADH2) is covalently attached to SDH, and transfers the
electrons through iron sulfur centers to ubiquinone and the electron transport system where their
free energy is used to generate ATP.
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The above reaction is difficult to detect directly because succinate and fumarate are not colored
molecules, and only small amounts of FAD are present. However, we can indirectly measure the
amount of FADH2 produced by coupling the above reaction to a second reaction. The reduced form
of the coenzyme (FADH2) transfers electrons to the electron transport system to be used in the
generation of ATP. The second reaction involves the use of an artificial electron acceptor, 2, 6dichlorophenolindophenol (DCIP) to accept electrons from FADH2. This acceptance of electrons
results in a corresponding color change from blue to colorless. Thus, the activity of succinate
dehydrogenase will be assayed by replacing the electron acceptor in the electron transport system
with an artificial electron acceptor whose reduction can be measured spectrophotometrically. To
ensure that electrons are passed to the dye and not funneled to the electron transport system, sodium
azide or potassium cyanide is added. This chemical inhibits the action of cytochrome oxidase, the
terminal component in the electron transport chain, and effectively bottlenecks the chain, thereby
preventing the acceptance of electrons from FADH2.
To summarize:
Rx. 1:
Rx. 2:

Succinate dehydrogenase
Succinate + FAD ----------------------------> fumarate + FADH2
FADH2 + DCIPox --------------------------> FAD + DCIPred
(blue)
(colorless)

Again, a successful reaction can be easily monitored using a spectrophotometer and will be
indicated by a decrease in OD units (measured as Abs620nm) over time.
Mitochondria will be isolated from cauliflower tissue by differential centrifugation. Upon
completion of this step, you will have samples of the original crude homogenate, the first pellet and
second (final) pellet and supernatant. Previous studies using other marker molecules and electron
microscopy indicate that the differential centrifugation steps outlined here will yield a concentration
of mitochondria in the second pellet; however this will not be monitored. The class or the lab
assistants will prepare a crude plant homogenate. Each group will receive a portion of this
homogenate for use in preparing a mitochondrial fraction by differential centrifugation. Each group
will then use this fraction to look for the presence of succinate dehydrogenase activity.
The activity of an enzyme varies greatly with conditions, with the purity of the samples, and with
the source of the enzyme. Therefore, any fair comparison of enzyme activity must compare all
test samples to the same standard scale. The International Union of Biochemistry has set a
standard for reporting enzyme activity called the Specific Activity (SA). It involves a measure
of the purity of the enzyme (amount of protein that has enzyme activity versus the total amount
of protein in the stock solution) and a measure of the amount of activity under specified, standard
conditions called the Enzyme Unit or EU. An EU is defined as the amount of enzyme necessary
to catalyze the formation of 1 micromole (1 x 10-6 moles) of product in one minute at 30 oC.
Specific Activity (SA) is defined as the number of Enzyme Units per milligram of total protein in
the stock solution. In your analysis, you will need to convert all your raw data to SA in order to
make good comparisons between your tests.

The prediction for this lab will be:

If the citric acid cycle occurs within the mitochondria then the fraction with mitochondria
will have the highest SA values.
So, if the fraction that contains the mitochondria has the greatest SDH activity it has the greatest
presence of the enzyme. Why might the other fractions contain SDH if it is normally found in the
mitochondrion?
A. Preparation of Cauliflower Homogenate
(THESE PROCEDURES WILL BE DONE FOR YOU BEFORE YOU COME TO LAB. Please familiarize yourself with the procedures
you will be responsible for them on your lab exam.)

Procedure:
Cauliflower heads have been obtained fresh from the grocery. Selected tissue will be
removed from the head and homogenized in a grinding buffer. The grinding medium is
composed of 0.5 M sucrose and 0.1 M KH2PO4, pH 7.3. PERFORM ALL
PROCEDURES IN AN ICE BATH TO PREVENT DETERIORATION OF THE
MATERIAL.
1.
2.

3.

4.

5.

Shave off the top 2-3 mm of the cauliflower head with a razor blade (2-3 heads
per 5 groups.
Weigh the sliced cauliflower. All weights and volumes in the instructions below
are based on approximately 25 g of tissue. If more cauliflower tissue is used
adjust the amounts accordingly. KEEP THIS AND ALL ISOLATION
MATERIALS ON ICE.
Place the cauliflower into a chilled blender. Add 20 ml of cold grinding buffer
and blend for about 10 seconds until a uniform slurry is achieved. Over blending
will not only rupture cells, but may damage mitochondria.
Pour the slurry through four layers of cheesecloth. Collect the filtrate in cold
polycarbonate centrifuge tubes. Wash the blender with 5-10 ml of cold grinding
buffer and pour the wash through the cheesecloth as well. Squeeze out all of the
juice into cold tubes. This slurry is the homogenate.
Transfer 2 ml of the crude homogenate to a clean tube and label as H. KEEP
THIS AND ALL SUBSEQUENT FRACTIONS ON ICE TO PRESERVE
MITOCHONDRIAL/ENZYME ACTIVITY.

B. Isolation of Cauliflower Mitochondria by Differential Centrifugation

(THESE PROCEDURES WILL BE DONE FOR YOU BEFORE YOU COME TO LAB. Please familiarize yourself with the procedures
you will be responsible for them on your lab exam.)

Procedure:
1.
Using the pre-chilled, high-speed refrigerated centrifuge, centrifuge the rest of the
homogenate at 500 x g for 10 minutes at 0 - 4 oC to remove large cellular debris.
The procedure for using the high speed, refrigerated centrifuge is as follows:
a.
Use the SS-34 rotor and polycarbonate tubes.
3

b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
2.

3.

4.
5.

6.

Make sure that the temperature of the rotor and the centrifuge is at 0 oC.
Remove the rotor cover by loosening the black screw and then the silver
screw.
Check for rubber stoppers at the bottom of each well to be used.
Place the weight-balanced tubes into wells directly opposite one another.
Replace the rotor cover by first tightening the silver screw and then the
black screw.
Close the door and hit the BRAKE button.
Set angular velocity for the correct rpm reading.
Set timer for the correct time.
Set rate = 0.
Hit START button.

Upon completion of this centrifugation step, you should have a visible pellet and
supernatant (containing mitochondria). Carefully transfer the supernatant (S1) to
a clean, cold centrifuge tube. Save for step 4.
Resuspend the pellet in 4 ml of cold suspending medium (NOT grinding buffer).
Label this sample/cellular fraction as P1 (the first pellet material). Store fraction
P1 on ice. It may be necessary to use this fraction later in the exercise.
Weigh balance the tube containing the supernatant (from step 2) and centrifuge at
20,000 x g for 30 minutes (0 - 4 oC) to pellet the mitochondria.
Decant the supernatant into a clean graduated centrifuge tube. It should contain
ribosomes, pieces of the endoplasmic reticulum, and other small or soluble
cellular components. Label this fraction S2. Save fraction S2 on ice. It may be
necessary to use this fraction later in the exercise.
The pellet should appear cream colored. Resuspend the mitochondrial pellet in 4
ml of suspending medium. Label this fraction P2. Save fraction P2 on ice.

Do not write on or place tape on the Cuvettes !!!!

C. Assay for Succinate Dehydrogenase

(THESE PROCEDURES ARE TO BE COMPLETED BY YOU IN LAB).
Procedure:
1. Set the wavelength of the spectrophotometer to 620 nm.
2. Designate two cuvettes 1 and 2. They will be prepared as follows:
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Hint: For tubes

with DCIP and
succinate, do not
add DCIP or
succinate until the
very last minute.

Phosphate buffer
MgSO4 (0.5 M)
KCN (0.1 M)
HCl (5%, v/v)
Water
Sodium succinate (0.25 M)
P2 suspension
DCIP
Total volume

Cuvette 1
1.0 ml
0.1
0.2
0.1
3.8
0.4
0.4
none
6.0 ml

Cuvette 2
1.0 ml
0.1
0.2
0.1
4.0
0.4
none
0.2
6.0 ml

Prepare Cuvette 1 first. Seal the cuvette with parafilm and then gently mix contents
by inverting cuvette. Use this to blank the spectrophotometer (follow the
instructions that are located on the spectrophotometer for achieving 0% absorbance).
Record the absorbance at 1 minute intervals for 15 minutes.
Prepare Cuvette 2. Mix contents. Place cuvette in spetrophotometer and record
absorbance in the data table provided. This is your time 0 recording for Tube 2.
Record the absorbance at 1 minute intervals for 15 minutes. Notice that the
contents of Tube 2 are identical to that of Tube 4, with the exception that the P2
suspension has been omitted. What is the purpose of this tube?
3. In the meantime, prepare the third and fourth cuvettes as follows:
Cuvette 3
Cuvette 4
Phosphate buffer
1.0 ml
1.0 ml
MgSO4 (0.5 M)
0.1
0.1
KCN (0.1 M)
0.2
0.2
HCl (5%, v/v)
0.1
0.1
Water
4.0
3.6
Sodium succinate (0.25 M) 0.0
0.4
P2 suspension
0.4
0.4
DCIP
0.2
0.2
Total volume
6.0 ml
6.0 ml
For cuvette 3: Mix contents. Place cuvette in spetrophotometer and record
absorbance in the data table provided. This is your time 0 recording for Tube 3.
Record the absorbance at 1 minute intervals for 15 minutes. Notice that the
contents of Tube 3 are identical to that of Tube 4, with the exception that the
sodium succinate has been omitted. What is the purpose of this tube?
For cuvette 4: NOTE: work quickly in preparing and using this cuvette.
If mitochondria are present and active (with working succinate dehydrogenase),
the reduction of DCIP will begin immediately upon the addition of sodium
succinate (the substrate). Mix contents. Place cuvette in spetrophotometer and
record the absorbance in the data table provided. This is your time 0 recording for
Tube 2. Record the absorbance at 1 minute intervals for 15 minutes.
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4. Now, prepare the fifth and sixth cuvettes as follows:

Cuvette 5
Phosphate buffer
1.0 ml
MgSO4 (0.5 M)
0.1
KCN (0.1 M)
0.2
HCl (5%, v/v)
0.1
Water
3.8
Sodium succinate (0.25 M) 0.4
P1 suspension
0.4
DCIP
none
Total volume
6.0 ml

Cuvette 6
1.0 ml
0.1
0.2
0.1
3.6
0.4
0.4
0.2
6.0 ml

Cuvette 5 is the blank for cuvette 6. Prepare this tube first and blank the
spetrophotometer before preparing Cuvette 6.
For cuvette 6: NOTE: work quickly in preparing and using this cuvette.
If mitochondria are present and active (with working succinate dehydrogenase),
the reduction of DCIP will begin immediately upon the addition of sodium
succinate (the substrate). Mix contents. Place cuvette in spetrophotometer and
record the absorbance in the data table provided. This is your time 0 recording for
Tube 6. Record the absorbance at 1 minute intervals for 15 minutes.
5. Finally, prepare the fifth and sixth cuvettes as follows:
Cuvette 7
Phosphate buffer
1.0 ml
MgSO4 (0.5 M)
0.1
KCN (0.1 M)
0.2
HCl (5%, v/v)
0.1
Water
3.8
Sodium succinate (0.25 M) 0.4
S2 suspension
0.4
DCIP
none
Total volume
6.0 ml

Cuvette 8
1.0 ml
0.1
0.2
0.1
3.6
0.4
0.4
0.2
6.0 ml

Cuvette 7 is the blank for cuvette 8. Prepare this tube first and blank the
spetrophotometer before preparing Cuvette 8.
For cuvette 8: NOTE: work quickly in preparing and using this cuvette.
If mitochondria are present and active (with working succinate dehydrogenase),
the reduction of DCIP will begin immediately upon the addition of sodium
succinate (the substrate). Mix contents. Place cuvette in spetrophotometer and
record the absorbance in the data table provided. This is your time 0 recording for
Tube 8. Record the absorbance at 1
minute intervals for 15 minutes.

Title: _____________________________________________________ Date: ___________

Time
(min)

Cuvette 1
OD
readings
(no DCIP)

Cuvette 2
OD
readings
(no P2)

Cuvette 3 OD
readings
(no succinate)

Cuvette 4
OD
readings
(P2)

Cuvette 5
OD
readings
(P1)

Cuvette 6
OD
readings
(S2)

0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Avg.
change in
OD/min

A. Determine the slope for the linear range of each plot. This is the average change in OD/min
or an estimate of reaction rate. These will be used in calculating SA.
B. Prepare a graph of OD and time (x-axis) for all fractions and solutions tested. All plots should
be placed on one graph.

C. Standardization Procedures: Enzyme Units and Specific Activity

Enzyme Units (How much enzyme activity was needed to completely catalyze each tube of
DCIP?)
The amount of AP'ase enzyme needed to convert 2 moles of PNPP into two moles of PNP should
be twice as much as the amount of AP'ase needed to convert 1 mole of PNPP into 1 mole of
PNP. This will be true PROVIDED ALL OTHER PHYSICAL CONDITIONS AND THE
TIME ARE CONSTANT. Therefore, it is always necessary to control the conditions under
which a reaction is taking place. In order to ensure uniformity in reporting results, the
International Union of Biochemistry has set up a standard based on the Enzyme Unit (EU). AN
EU IS DEFINED AS THE AMOUNT OF ENZYME NECESSARY TO CATALYZE THE
FORMATION OF 1 MICROMOLE (1 X 10-6 MOLES) OF PRODUCT IN ONE MINUTE AT
30 oC.
Consider the following steps for calculating EU:
1.

The absorbency of one mole of DCIP under standard conditions has been reported as 19.1
OD units. If this is accurate, then the absorbency of one micromole of PNP under
standard conditions corresponds with a change of __________________ OD units.

2.

Since 1 EU of enzyme catalyzes one micromole of DCIP under standard conditions, then
1 EU must also correspond with a change of ___________ OD units (this is a very small
number, which will result in a large number in Step 5).

3.

The average OD increase per minute is calculated as:

OD at 15 minutes - OD at 0 minutes
15 minutes

4.

The total dilution of the enzyme is determined as follows:

DILUTION FACTOR FROM ADDING SUBSTRATE

(Step 7 of spec readings)
5.

EU can now be calculated as:

Average OD/minute
(from Step 3)
x
OD/micromoles DCIP
(from Step 2)

Total Dilution =
(from Step 4)

micromoles catalyzed DCIP = ______ EU

minute

Specific Activity (How much enzyme activity is in each mg of enzyme solution?)

Once an enzyme is extracted from a specific tissue or cell type, the complete separation of that
enzyme from all other proteins is a difficult and often unnecessary task. Usually the total
amount of protein in a cell extract is much greater than the amount of enzyme present. Since the
relative purity of an enzyme extract is often critical to researchers, the International Union of
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Biochemistry has established a measurement of purity called the Specific Activity (SA). THE
SA IS DEFINED AS THE NUMBER OF ENZYME UNITS (EU) PER MILLIGRAM OF
TOTAL PROTEIN IN THE SAMPLE. In order to calculate SA, the concentration of total
protein (mg/ml) in the original fraction must be determined. In Laboratory 3, the method for
determining total protein was introduced -- the Biuret test.
Use the protocol for the Biuret test in Laboratory #3 and the Standard Curve of Protein
Concentration to determine the concentration of total protein in the solution.
TUBE 1:
TUBE 2:
TUBE 3:
TUBE 4:

4.5 ml of Biuret reagent + 0.5 ml distilled water

4.5 ml of Biuret reagent + 0.5 ml of P1
4.5 ml of Biuret reagent + 0.5 ml of P2
4.5 ml of Biuret reagent + 0.5 ml of S2

Use the directions from Lab #3 to complete this test.

What is the dilution factor for the protein at this step? ______________

Fraction

OD Reading

Protein
Concentration from
Graph

Concentration
corrected for
dilution
(multiply by df)

P1
P2
S2

D. Calculation of Enzyme Units and Specific Activities for This Data

Fraction

Average
OD/minute

OD/micromoles
DCIP

Total
Dilution

EU

SA = EU/
total protein
concentration
(from above)

P1
P2
S2