In 1937, Robert Hill discovered that isolated chloroplasts can generate oxygen when they are illuminated in the presence of a suitable electron acceptor, even in the absence of carbon dioxide. This finding was a landmark in the study of photosynthesis because it established that the source of the electrons used in the light reactions is water. It also confirmed that the released oxygen is derived from water instead of carbon dioxide. In chloroplasts, the final electron acceptor is NADP+, which is reduced to NADPH.
ATP is generated by photophosphorylation during the light-induced electron transfer reaction. NADPH and ATP produced in the light reactions are utilized for the biosynthetic reactions in the dark reactions for making carbohydrates.
Light reactions can be investigated in the laboratory using isolated chloroplasts. The Hill reaction is defined as the reduction of an electron acceptor (A) by electrons from water, with the evolution of oxygen, when isolated chloroplasts are exposed to light:
The reagent 2,6-dichlorophenolindophenol (DCPIP) is a useful artificial electron acceptor.
The following reaction can be monitored by measuring the loss of the blue color of DCPIP. DCPIP replaces NADP as the final electron acceptor in the light reactions.
Light reactions can be affected by the presence of different compounds. The herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; known commercially as diuron) inhibits the transport of electrons from PQA to PQB. On the other hand, ammonia eliminates the H+ gradient across the thylakoid membrane, thus functioning as an uncoupler of electron transport and photophosphorylation. There are three objectives for this laboratory.
(1) To isolate active chloroplasts
from spinach leaves. (2) To measure the rate of the Hill reaction. (3) To examine the effects of DCUM and NH4OH on the rate of the Hill reaction. 1
1. Chloroplast Isolation
The isolation procedure will be performed at 0-4 C (on ice). Pre-chilled solutions and containers will be used. Grind about 5 g of Chinese spinach leaves (midribs and petioles removed) in a mortar using 10 ml of phosphate extraction medium (0.05 M phosphate buffer, pH 7.0; 0.4 M sucrose; 0.01 M KCl). Add more extraction buffer up to 40 ml. Afterwards, filter the extract through 2 layers of cheesecloth and centrifuge at 1,000 × g. Pour off the supernatant and resuspend the chloroplast pellet in 10 ml of extraction medium.
chilled on ice.
Keep the chloroplast suspension
2. Measurement of Hill reaction
Turn on the spectrophotometer at least 5 min before taking the readings.
ml of the chloroplast suspension and boil it in a heat block (95 C) for 1 min. Then, label five cuvettes (1 to 5). Cover cuvette 2 with aluminum foil. Pipette 1 ml of the reagent (DCPIP in phosphate buffer) to each cuvette.
Prepare the following reaction. Chloroplast suspension should be added LAST. For cuvette 3, use boiled chloroplast suspension.
(10-4M) (0.01 N)
17 µl (boiled) 100 µl
After adding the chloroplast suspension, mix the contents in the cuvettes and measure the absorbance at 600 nm.
Use 1 ml phosphate buffer plus boiled
chloroplast suspension as the blank.
For cuvettes 1, 3, 4, and 5, place them ~30 cm away from a light bulb and take absorbance readings every minute for the first 5 min, and then every 5 min for another 25 min. Remove the aluminum foil from cuvette 2 and measure the absorbance at the end of the experiment.
Lab Report (Email the report to your demonstrator before 23:59 this Sunday) 1. Plot all your data in ONE graph
2. What are the purposes for cuvettes 2 and 3? What conclusion can you make about the absorbance change in cuvette 1? Explain your answers. 3. Shown below is the electron flow in the two photosystems. DCMU interferes with the electron transfer from PQA to PQB. DCPIP accepts electrons from PQB. (a) What conclusion can you make on the absorbance change in cuvette 4? Explain your answer.
(b) If DCPIP accepts electrons from Ph a, would you still see the same absorbance change? Why?
4. Protons are pumped to the thylakoid lumen during electron transfer through the Cyt bf complex. However, ammonia eliminates the proton gradient across the thylakoid membrane. Explain the absorbance change in cuvette 5. 5. In cuvette 1, would you expect carbohydrate synthesis to occur during the decoloration process? Explain your answer.
6. Using the available resources in the lab session today, design an experiment to investigate the effect of light intensity on the Hill reaction.
Cite this Hill Reaction Essay
Hill Reaction Essay. (2017, Feb 06). Retrieved from https://graduateway.com/hill-reaction/