#7 Exercise Pre-lab
You may submit one pre-lab for the group
Names of Group Members
______________________
_______________________
_______________________
Section number___________________
TA_____________________________
Date____________________________
1. (10 points) Present the hypothesis you will be testing and describe what you will expect to see
if your hypothesis is correct.
We will be performing an experiment to determine whether malonate is a competitive or
noncompetitive inhibitor of succinate dehydrogenase. Based on our previous experiments it is
difficult to predict if malonate is a competitive or noncompetitive inhibitor. We therefore
arbitrarily hypothesize that malonate is a competitive inhibitor and that it competes with the
substrate for the active site on the enzyme, succinate dehydrogenase. We then expect to see a
higher Km with the inhibitor present and the same Vmax. If the inhibitor is a noncompetitive
inhibitor, we would expect to see a lower Vmax and no change in the Km value. To perform this
experiment we will have both a control and experimental group. In the control group the amount
of substrate varies and no inhibitor is present. In the experimental group, the amount of susbtrate
varies and a constant amount, 0.5 mL, of inhibitor is present for each reaction. By comparing the
control and experimental groups and plotting the data on a Lineweaver-Burke plot to determine
the Km and Vmax for each group, we can determine whether malonate is a competitive or
noncompetitive inhibitor.
2. (15 points) Provide a flow chart that describes how you will conduct the experiment. Be sure
to include appropriate controls. (Note here: If it is relevant to your experiment to run a control
series that is like one that you did the previous week, be sure to repeat that series. You cannot
compare the results of two different mitochondrial preparations directly).
Prepare Intact Mitochondria
1. Cut 20g off outer the portion of the cauliflower. Keep the tissue on ice while collecting.
2. Grind the 20 g of cauliflower with 40 mL of ice cold mannitol grinding solution and 5g of
sand in a chilled mortar.
3. Filter the cauliflower through 4 layers of cheesecloth into a centrifuge tube.
4. Centrifuge for 10 min at 4 degrees Celsius at 2500 rpm.
5. Pour off the liquid (supernatant) into a new centrifuge tube and centrifuge that for 30 min at 4
degrees and at 10000 rpm.
6. Pour off the supernatant , add 7 mL of ice cold assay medium to the pellet, resuspend the
pellet, keep on ice.
7. Turn on spectrometer and set absorbance to 600 nm.
8. Prepare 12 tubes, the first 6 will serve as the control group and the second group of 6 tubes
will serve as the experimental group. Dilutions will need to be made of the succinate. Use the
table below to perform the dilutions.
Succinate
mL
Tube
A
B
C
D
E
5(A)
5(B)
5 (C)
.8 (D)
Concentration
Diluent mL mM
5
0
200
5
100
5
50
5
25
9.2
2
Control Group
Tube
Blank
1
2
3
4
5
Assay Medium
Azide
DCPIP
Succinate
mL
mL
mL
mL
3.5
0.5
0 0.5 A
3
0.5
0.5 0.5 A
3
0.5
0.5 0.5 B
3
0.5
0.5 0 .5 C
3
0.5
0.5 0.5 D
3
0.5
0.5 0.5 E
Mitochondria
mL
0.5
0.5
0.5
0.5
0.5
0.5
Experimental Group
Tube
Blank
I1
I2
I3
I4
I5
Assay Medium
mL
3
2.5
2.5
2.5
2.5
2.5
Azide
DCPIP
Succinate
mL
mL
mL
0.5
0 0.5 A
0.5
0.5 0.5 A
0.5
0.5 0.5 B
0.5
0.5 0.5 C
0.5
0.5 0.5 D
0.5
0.5 0.5E
Mitochondria
Inhibitor
mL
mL
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
9. For each tube pipet all of the solutions except the mitochondrial suspensions. Mix by
inverting the tubes twice.
10. To begin each reaction, add the mitochondria, mix and then take the zero reading in the
spectrometer. Take readings every 5 min for a 20 min period. Start each group by placing the
blank tube in the machine, to zero the spectrometer. Also, between each set of reading, place the
blank in the spectrometer and rezero the machine if necessary.