Hill Reaction

Question 1

Define Hill Reaction

Answer 1

They are a phase of light reactions in photosynthesis. It is the light driven transfer of electrons from water to Hill Reagents. A colour change after adding the Hill Reagents allows you to monitor the reaction.

Question 2

What are Hill Reagents?

Answer 2

They are electron acceptors supplied externally by researchers.

Question 3

Purpose of the stomata in photosynthesis

Answer 3

CO2 enters and O2 exits the leaf through stomata, which are microscopic pores in leaves.

Question 4

Purpose of thylakoids in photosynthesis

Answer 4

The chlorophyll is in the membranes of the thylakoids, which are connected sacs in the chloroplast. They are stacked in columns called grana.

Question 5

What are stoma

Answer 5

Located in chloroplasts, which is a dense interior fluid.

Question 6

What are the two stages of photosynthesis and where does each occur? Which occurs first?

Answer 6

1. light reactions- thylakoid membrane, the photo part
2. Calvin cycle- cytosol, the synthesis part

The light reactions occur first.

Question 7

What is the purpose of the light reactions? Explain the light reactions in terms of photosystems, and what the produces of the light reactions are ultimately used for.

Answer 7

• split H20
• generate NADP+ from NADPH
• Generate ATP from ADP through photophosphorylation
• release O2

The light that hits p680 photosystem excites electrons and transfers them to p700 photosystem through ETC. The p680 electrons are replaced by electrons generated when water splits. The high energy electrons to p700 drives ATP production from chemiosmosis. The electrons of p700 are photoexcited to a second electron transport chain and used to reduce NADP+ to NADPH. The ATP and NADPH is used as chemical energy to fix CO2 into sugars in the calvin cycle.

Question 8

Where are the electron transport molecules located?

Answer 8

The membrane lumen of the thylakoids.

Question 9

What are the two photosystems in the thylakoid membrane, what are their functions, which occurs first?

Answer 9

Photosystem II and Photosystem I. Photosystem II occurs first, and it absorbs 680nm wavelengths. Photosystem I absorbs a wavelength of 700nm.

Question 10

Where does the electron transport chain occur?

Answer 10

Lumen of thylakoids

Question 11

Why do you use DCMU, what reaction does DCMU stop? What are the repercussions of stopping this reaction?

Answer 11

Use DCMU to stop the flow of electrons from PQ –> PQH2 in the electron transport chain from the p680 photosystem. Do this to control where the electrons go. Stopping this reaction stops ATP synthesis

Question 12

What is the function of NH3?

Answer 12

It inhibits the Hill reaction. It uncouples the ETC from ATP synthesis by disrupting the proton gradient across the thylakoid membrane.

Question 13

What is DCIP and what is its function? Why do you want to use DCIP?

Answer 13

DCIP is a Hill Reagent that substitutes for NDAP during photosynthesis, and allows you to detect if there is a reduction. When it is oxidized before the reaction, it is blue. After it is reduced, post-photosynthesis, it turns white. You want to use it to measure the change in absorbance as DCIP is reduced, which indicates that photosynthesis is occurring, and telling you that there is electron flow occurring.

Question 14

What is the purpose of this lab?

Answer 14

Learn how to isolate organelles through centrifugation, measure the rate of the Hill reaction in vitro using isolated chloroplasts. See how different inhibitors affect the rate of the Hill reaction.

Question 15

What is cellular fractionation? What are the two types of cellular fractionation?

Answer 15

Isolation of subcellular organelles. Two types of cellular fractionation is density gradient centrifugation and differential centrifugation.

Question 16

What is density gradient centrifugation?

Answer 16

Put a cellular homogenate to a tube with medium with increasing density going down the tube. After centrifugation, the particles settle where the gradient is equal to their density.

Question 17

What is differential centrifugation?

Answer 17

Prepare a tissue homogenate and centrifuge it at successively higher speeds to sediment progressively smaller particles. The largest things experience a greater centripital force, and move more rapidly to the bottom so they require spinning at lower speeds.

Question 18

How do you calculate the relative centrifugal force a preparation is subjected to?

Answer 18

RCF=1.119 * 10^5 (rpm)^2x

rpm=revolutions per minute
x=distance from axis of rotation to end of centrifuge tube

Question 19

What is photosynthesis? What is the equation of photosynthesis? Where does photosynthesis take place?

Answer 19

conversion of light energy into the chemical energy of sugars and other organic molecules.

6CO2 + 6H2O –light energy –> C6H12O6 + 6O2

It takes place in the chloroplasts of leaves.

Question 20

How do you calculate the total change in absorbance for each time interval?

Answer 20

ChangeA=A0min-Axmin

Question 21

Explain Atrazine

Answer 21

In post lab

Question 22

Why do you completely resuspend the pellet after the second centrifugation in differential centrifugation?

Answer 22

You want equal number of chloroplasts in each tube. If you have unequal number of chloroplasts, you would incorrectly measure the rate of photosynthesis and get a false higher or false lower reading.

Question 23

Why is the blank tube important?

Answer 23

It is to blank the instrument, to tell the instrument that it is zero to set a baseline to compare the rest of the measurements. Adding chloroplasts to the blank so the only change in absorbance you see is the change in DCIP colour.

Question 24

Why can you do the hill reaction in water?

Answer 24

Water keeps the temperature more even than air.

Question 25

How did you make the control tube?

Answer 25

You wrapped it in foil, so it does not get light, so photosynthesis does not occur.

Question 26

Initially, what was the hypothesis that CO2 was split during the light reactions?

Answer 26

CO2

Question 27

How did they know that H2O was split during the light reactions?

Answer 27

Bacteria that used H2S rather than water did not produce oxygen as a photosynthetic byproduct, but sulfur instead. Then Hill showed that chloroplasts released oxygen even in the absence of CO2. Thus, the source of oxygen should be H2O because an electron acceptor is photoreduced by the hydrogens and electrons of water. This process is called the Hill reaction.

Question 28

How was the Hill reaction confirmed?

Answer 28

It was confirmed using a radioactive isotope of oxygen.

Question 29

How do we measure the rate of the Hill reaction in isolated chloroplasts in this lab?

Answer 29

We use DCIP (artificial electron acceptor) which turns colourless from blue when it is reduced.