Lab 4

Question 1

citric acid cycle

Answer 1

generating the reduced electron carriers which funnel into the reactions of the electron transport chain and oxidative phosphorylation (or ATP synthesis) which are embedded in the inner membrane

Question 2

succinate dehydrogenase location/coenxyme

Answer 2

FAD are tightly bound to inner membrane (catalyses oxidation of succinate to fumerate)

Question 3

succinate replaced with what?

Answer 3

DCIP (2,6-dichlorophenolindophenol) Oxidized dye is blue, reduced is clear

Question 4

What inhances DCIP more?

Answer 4

inhibiting the electron transport chain (its now the only carrier so electrons go here) sodium azide (blocks final transfer of e- from cyt oxidase to O2)

Question 5

Malonate is what?

Answer 5

competoitive inhibiotr (cant be oxidized or dehydrogenized)

Question 6

Assuming that a mM of DCIP is reduced for each mM of succinate oxidized by succinate dehydrogenase, calculate the maximum observed rate of enzyme activity
(expressed as mM of succinate oxidized/minute) for each experimental preparation
(show your calculations).
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Answer 6

From the standard curve, the graph equation was:
y = 0.0188x + 0.005
where y was the absorbance and x was the concentration in μM

For line 10:

Starting absorbance
0.394=0.0188x + 0.005
x=15.9 μM of succinate oxidized

Ending absorbance
0.422=0.0188x + 0.005
x=22.2 μM of succinate oxidized

22.2-15.9=6.3 μM of succinate oxidized in 30 minutes
=0.209 μM of succinate oxidized/min

Question 7

Azide is a respiratory poison that blocks electron transport at the level of cytochrome oxidase in the respiratory chain. What effect would the omission of azide have on the rate of dye reduction in this experiment? Explain.

Answer 7

Azide block the last transfer of electrons from cytochrome oxidase to oxygen, which therefore blocks the entire chain. If azide was not added, the electrons would be reduced by other aspects of the electron chain as well, and the activity in DCIP would be less pronounced. Since DCIP would not be the only electron acceptor, the electrons would have another option and would go to DCIP less and more consistent.

Question 8

The contents of each tube directly affects how much enzyme activity is recorded. Therefore, once you have identified each tube, explain the contents and their affect on the enzyme activity observed.

Answer 8

Test tube 2 (C) contains the normal amount of mitochondrial pellet, 0.3ml. The graph shows that the rate of absorbance does decrease over time, but it’s not a very pronounced decrease as there is not a lot of mitochondria sample to work with, so the DCIP can’t get reduced as much. Test tube 6 (D) has twice the amount of mitochondrial pellet in it then 2 had, with 0.6mL. This means there is more mitochondria sample, so more succinate dehydrogenase for the DCIP to react with and therefore more DCIP reduced (and more succinate oxidized). Test tube 4 (B) had triple the amount of mitochondrial pellet then 2 did with 0.9mL. This means there was even more mitochondrial to react with so there’s even more of a decrease in the rate. Finally, tube 10 (A) had the boiling mitochondrial pellet which has a very flat line. This means there is really no change in activity which makes sense since the enzyme won’t work, because boiling it cause it to get denatured.