One of the first steps of photosynthesis is the splitting of water to donate two electrons to the reaction center, P680. This reaction is known as the Hill reaction. The electrons donated to P680 move through an electron transport chain to the reaction center P700, and eventually, to reduce NADP to NADPH. The complete reaction can be summarized as follows:
Other electron acceptors can be substituted for NADP, which allow the Hill reaction to be measured, and some of it components studied. The most commonly used electron acceptor is the dye 2,6 dichlorophenolindophenol (DCIP), which can accept electrons instead of P700. When DCIP is added to a chloroplast or thylakoid suspension, the following reaction occurs:
DCIP is a blue color in its oxidized form. When reduced, it is colorless. This property of DCIP allows the measurement of the rate of the Hill reaction.
Spinach chloroplasts have been prepared by blending 25 g of fresh leaves in l00 ml of a solution containing 0.3 M sucrose and l25 mM potassium phosphate buffer to maintain the pH at 7.5. The homogenate was filtered, and the filtrate centrifuged for 5 min at l000g. The pelleted chloroplasts were then stored on ice.
Procedures
1. Label 5 test tubes (1-5) with a marker. Prepare each tube with
the proper amount (ml) of chloroplasts, water, and dye (DCIP) as described
below.
| Tube # | Chloroplasts | Buffer | Water | DCIP |
| 1 | 0.5 | 3 | 1.5 | 0 |
| 2 | 0.5 | 3 | 0.5 | 1 |
| 3 | 0 | 3 | 1 | 1 |
| 4 * | 0.5 | 3 | 0.5 | 1 |
| 5 ** | 0.5 | 3 | 0.5 | 1 |
* After the initial absorbance reading, wrap this tube in aluminum foil.
** After the initial absorbance reading, add 8 drops of DCMU to this
tube.
2. Record the initial absorbance of each tube (at 550 nm), then record the absorbance after 5 minute intervals for 25 minutes.
3. Design a table to record your results.
Write up a brief report on this lab exercise. Include the following information:
· Materials and methods
· Explanation of the Hill reaction
· Graph of absorbances for all tubes
· Explanation of results (why did the absorbances fluctuate
as they did?)
· Answers to the following questions
Where in the light reactions must DCMU be acting?
What would you observe if the following were done?
a) water splitting inhibited
b) P700 destroyed
c) electron transport uncoupled from photophosphorylation.