Hill Reaction

Lab 8

THE HILL REACTION OF PHOTOSYNTHESIS

I. Introduction

Photosynthetic organisms are able to capture light energy and use it to form carbohydrates and free oxygen. The overall equation for the photosynthetic reaction is as follows:

2,000 Kcal light energy

plants and algae

6 CO₂ + 12 H₂O -------------------------------------> C₆H₁₂O₆ + 6 O₂ + 6 H₂O

chlorophyll

bacterial - H₂, H₂S, NH₃

It is important to note that plants and algae use water as their source of electrons but bacteria may use H₂, H₂S or NH₃ as a source of electrons. photosynthesis occurs in large organelles called chloroplasts. The first stage involves the light reactions where light energy is trapped by the chloroplast and results in the phosphorylation of ADP from ATP and the reduction of another energy containing molecule NADP⁺. The light dependant reactions of photosynthesis are outlined below.

ferredoxin

(electron acceptor) 2 e-

NADP+

NADPH

(electron Acceptor) e-

h e-

i e-

ADP m

o e-

e- s

ATP i

Photosystem I

(P₇₀₀)

e 2H⁺ 2 O₂

Photosystem II

(P₆₈₀)

H₂O

We are interested in the ability of chlorophyll-a in the reaction center of PSII₆₈₀ to split water and liberate free molecular oxygen in the process. In 1937 Robert Hill showed that isolated chloroplasts can evolve oxygen in the absence of CO₂. His finding was one of the first indications that the source of the electrons in the light reactions was in fact water. In his in vitro system, He provided an artificial electron acceptor. The artificial electron acceptor intercepts the electrons before they cascade down to PSI₇₀₀, but after they have gone down the electron transport chain. Various dyes can be used as the artificial electron acceptor (A) so that the general equation, known as the Hill Reaction can be written as follows:

(blue) (colorless)

H₂O + A ----------------------> AH₂ + 1/2O₂

The Hill reaction is formally defined as the photoreduction of an electron acceptor by the hydrogens of water, with the evolution of oxygen. In vivo, or in the organism the final electron acceptor is NADP⁺. We can measure the rate of the Hill reaction in isolated chloroplasts. This procedure uses a dye as an artificial electron acceptor that changes color as it is reduced. DCIP (2,6-dichlorophenolindophenol) is a dye which is blue in its oxidized form and colorless in its reduced form.

The change in absorbance (at 600nm) will be used to measure the rate of the Hill reaction. The change in absorbance will be measured at 1 minute intervals of exposure to an intense light source. Since the DCIP will begin to revert to its oxidized (blue) state as soon as the chloroplasts in the reaction vessel are removed from the light path, it is essential that all absorbance readings be taken as quickly as possible.

II. Procedures

You will obtain the chloroplasts from spinach leaves using a modification of a standard fractionation procedure described by Whaltey and Arnon (1962). Start by homogenizing the spinach in a mortar with a pestle with buffered, isotonic salt solution and a small quantity of sand. After squeezing the homogenate through cheesecloth to remove the larger pieces, you will centrifuge the filtrate at 200 g for 1 minute, sedimenting the debris and whole cells. You will then centrifuge the supernatant at 1,300 g for 5 minutes, sedimenting most of the chloroplasts and nuclei. For all cell fractionation procedures, the solutions and containers must be maintained at 0^oC.

1. Weigh out 4 gm of fresh spinach leaves from which the major veins have been removed, rinse and blot dry.

2. Cut the leaves into small pieces with scissors and place in a chilled mortar with 15 ml of ice-cold Tris-NaCl buffer and a sprinkling of purified sand. Grind the tissue with a chilled pestle for 2 minutes.

3. Filter the suspension through four layers of cheesecloth into a chilled 15 ml conical centrifuge tube. Also wring out the juice into the centrifuge tube.

4. Centrifuge the filtrate at 1,400 RPM with a 10 cm rotor for 1 minute. Make sure that the centrifuge tubes are balanced, that is the tubes are opposite each other should have the same total volume.

5. Decant the supernatant into a clean, chilled centrifuge tube and spin at 3,500 RPM with a 10 cm rotor for 5 minutes, remember to balance the tubes.

6. Decant and discard the supernatant and then, using a graduated cylinder, add 10 ml of ice-cold Tris-NaCl buffer to the pellet in the centrifuge tube. With a pasture pipet, thoroughly resuspend the pellet. To ensure that the chloroplast suspension is thoroughly mixed, cover the centrifuge tube with parafilm and invert several times.

7. Transfer 4.0 ml of the chloroplast suspension to a clean, chilled centrifuge tube and dilute with 6.0 ml of ice-cold Tris-NaCl buffer. Cover the test tube, invert several times, and place in an ice-water bath in which it should remain during the entire experiment. It is the diluted chloroplast suspension (we will call this the chloroplast stock suspension) that will be used to measure the Hill reaction.

8. Allow the spectrophotometer to warm up for at least 15 min. Set the wavelength for 600 nm.

9. Prepare a water bath as follows. Add 150 ml of water at 20C to a 250 ml beaker. During the illumination, the cuvettes are kept in the water bath to keep the reaction temperature fairly constant. Adjust the temperature of the water bath at the beginning of each experimental trial.

10. Place the water bath 25 cm from a lamp with a 100 watt frosted incandescent bulb. But do not turn on the lamp until the start of each experimental trial.

11. Label 3 cuvettes as shown in the following table. Except for the ice-cold chloroplast stock suspension, all solutions should be at room temperature.

12. Tube number 2* will act as a nonilluminated control. It will be wrapped with two layers of aluminum foil and loose fitting cap of foil for the top of the cuvette.

13. Prepare the reference blank and tube 2. Add the solutions in the sequence given across the top of the table, from the left to the right. Before and after adding the chloroplast suspension, which should be inverted several times just before it is added, cover each cuvette with parafilm and invert twice to mix the contents. Remove the parafilm and cover tube 2 with the foil cap. Note the time and set tube 2 aside. An absorbance reading should be taken after 15 min but, meanwhile, proceed with the steps that follow.

15. Prepare tube 3. Add the solutions in the same manner used for tube 2 in step 13. Immediately take the 0-min absorbance reading and record that value.

16. Immediately place tube 3 in the water bath, turn on the lamp, and note the time. After 1 min of illumination, remove the tube from the water bath, and quickly wipe the surface of the cuvette and measure its absorbance. All absorbance readings must be made quickly as possible.

17. Return tube 3 to the water bath and take absorbance readings at 1-min intervals of illumination for 15 minutes. Be sure to accurately record your readings. The spectrophotometer should be adjusted for the blank after every few readings (this would not be necessary on some of the better instruments). Remember to take the 15 min reading of tube 2. The absorbance should reach 0 in about 10 minutes.

18. Graph the rate of the Hill reaction as follows: Plot the total change in absorbance (ordinate) versus time (abscissa). Include the origin as a point for all curves, e.i. the 0-min value for the change in absorbance is assumed to be 0. Fit the curve using the options in Sigma Plot.

12. 200 gms of fresh spinach, just before use, rinse with cold tap water and blot dry

1. Bregman, A. 1990. Laboratory Investigations in Cell and Molecular Biology. Third Edition, John Wiley & Sons, New York.

2. Gasque, C.E. 1989. A Manual of Laboratory Experiences in Cell Biology. W.B. Brown, Dubuque Iowa.

3. Whatley, F.R. and Arnon, D.I. 1962. Photosynthetic phosphorylation in Plants. In Methods in Enzymology, Vol. E, Colowick, S.P. and Kaplan, N.O., Eds. PP 308-313. Academic Press. N.Y.

Design and perform an experiment to determine the effect of light intensity and inhibitors on the Hill Reaction. The two inhibitors you will use (use both of them in your experimental design) are ammonia and DCMU {3-(3,4-dichlorophenol)-1,1-dimethylurea}. Ammonia functions as an uncoupler or a compound that separates the process of phosphorylation from electron transport.

As a point of interest, the dye DCIP can itself be an effective uncoupler but at the low concentration we use in the present study there is still partial coupling so that the effects of other uncouplers can still be measured. The second compound you will study is DCMU which is a herbicide. It functions to block both electron transport and phosphorylation by interrupting electron flow at the beginning of the major electron transport chain. When designing an experiment to study the effects of light intensity on the Hill Reaction, keep in mind that the light intensity decreases as the square of the distance from the source.

12. 200 gms of fresh spinach, just before use, rinse with cold tap water and blot dry