Lab 7

Question 1

Explain the changes in pH that you observed: with the light off

Answer 1

With the light off, the pH of the solution decreased dramatically and then levelled off. This is because the electron transport chain stopped working, no photons are being brought in, so the protons that were in the thylakoid sacs are being pumped back into the storm by ATP synthase. This loss of hydrogen ions lowers the acidity of the thylakoid, and the pH of the solution goes down.

Question 2

Explain the changes in pH that you observed: with the light on

Answer 2

With the light on, the pH of the solution increased dramatically and then levelled off. This is because hydrogen ions are being continuously pumped from the storma to the thylakoid membrane. This increase in hydrogen ions accounts for the increase in pH we observed as, as the hydrogen ions cause the thylakoid sacs to be more acidic.

Question 3

TRUE?FALSE?
a) Light promotes photosynthetic electron transport in the thylakoid
membranes of chloroplasts, which causes protons to be actively translocated out
of the lumen of the thylakoids into the chloroplast stroma.
b) As the proton gradient increases, ADP synthesis is driven by the proton motive force thus generated.
c) In mitochondria, the transport of protons by the electron transport chain is from the intermembrane space into the matrix.

Answer 3

a) Light promotes photosynthetic electron transport in the thylakoid membranes of chloroplasts, which causes protons to be actively translocated out of the stroma of the thylakoids into the chloroplast lumen.
b) FALSE As the proton gradient increases, ATP synthesis is driven by the proton motive force thus generated.
c) FALSE In mitochondria, the transport of protons by the electron transport chain is from the matrix into the intermembrane space.

Question 4

During the first part of the procedure for this experiment (isolating chloroplasts from spinach leaves), you added a salt solution to the chloroplasts. What was the purpose of this step? If you were to leave this step out, how would it affect your results? Explain

Answer 4

The NaCl is added to swell the chloroplast making them for pH changes in the stoma to be detected and to protect against osmotic shock, which is substrates or any solute in the supernatant water is drawn out of the cells through osmosis. If we didn’t add NaCl, we couldn’t detect a change in pH in the external medium and we would assume nothing was happening, this is because the pH change would remain in the chloroplast and never make it to the outside medium.

Question 5

Explain the result obtained with the proton ionophore, CCCP.

Answer 5

.the results of adding the CCCP to the solution was very similar to when we covered the solutions. The pH dropped dramatically, lower then the previous two trials, and then levelled out at about 6.4

Question 6

In mitochondria, oxygen consumption and ATP production are normally
tightly coupled. How would the use of CCCP affect this relationship in
mitochondria?

Answer 6

CCCP is an uncoupler, it uncouples the formation of ATP from the consumption of oxygen which formed the hydrogen gradient. It enters and discharges the pH gradient, thus getting rid of the membrane potential. Without the pH gradient and the membrane potential, hydrogen ion have no reason to enter back into the cell through ATP synthase so ATP stops being produced. Protonophores are generally aromatic compounds with a negative charge, that are both hydrophobic and capable of distributing the negative charge over a number of atoms by π-orbitals which delocalize a proton’s charge when it attaches to the molecule.