NAME: LAB PARTNER:

ID#: DATE: Wednesday 6th October, 2010

Course Code: BIOL 2363 – Metabolism

Title of Lab: The Hill Reaction in Isolated Chloroplasts

Aim: To determine the chlorophyll content in 10g of grounded spinach leaves using the equation C=A/(ε x l); To investigate the effect of light intensity on the rate of the hill reaction and to investigate the effect of the inhibitors DCMU and ammonia on the rate of the hill reaction using DCPIP as an artificial electron acceptor and spectrophotometry absorbance readings at 600nm.

Theory:

Photosynthesis is the process by which solar energy is trapped and used to drive the synthesis of carbohydrate from carbon dioxide and water. It occurs in green plants, algae and photosynthetic bacteria (Hames and Hooper 2005).

6CO2 + 12H2O C6H12O6 + 6O2 + 6H2O

In green plants and algae, photosynthesis takes place in the chloroplasts.

(Gupta 2010)

The membrane system is the site of the light-dependant reaction. It is covered with chlorophyll and other pigments, enzymes and electron carriers. The light-independent reaction takes place in the stroma (Taylor, Green and Stout 1997).

In the light-dependant reaction, energy from light and hydrogen supplied by water is used to make ATP and NADPH. The ATP and NADPH products are used to carry out reactions in the light-independent stage to produce carbohydrate (Nelson and Cox 2008).

The Hill Reaction is the portion of the light reactions in which electrons from water are transferred to an electron acceptor, reducing the acceptor. This established that the source of electrons used in the light reactions was indeed from water (Lodish, et al. 2007).

In chloroplasts, the final electron acceptor is NADP+ which is reduced to form NADPH.

2 H2O + 2NADP+ + (light, chloroplasts) → 2 NADPH2 + O2

Fig 1: Chloroplast Structure

Fig. 2 : Effect of Light intensity on rate of photosynthesis (Taylor, Green and Stout 1997).

However, in this experiment, an artificial electron acceptor DCPIP was used. DCPIP changes colour from blue to colourless as it is reduced and hence the change in absorbance of this dye makes the Hill Reaction measurable.

2 H2O + 2DCPIP + (light, chloroplasts) → 2DCPIPH2 + O2

(BLUE) (COLOURLESS)

The rate of photosynthesis is an important factor in crop production since it affects yields and the rate of a biochemical process like photosynthesis will theoretically be limited by the slowest reaction in the series (Taylor, Green and Stout 1997).

The limiting factors looked at in this experiment are the effect of light intensity and the effect of inhibitors on the rate of the Hill Reaction.

Herbicides kill plants by inhibiting photosynthesis. The inhibitor DCMU interrupts the electron transport chain in photosynthesis and hence blocks the ability of the plant to turn light energy into chemical energy ATP (Taylor, Green and Stout 1997).

Ammonia eliminates the H+ gradient across the thylakoid membrane and therefore functions as an uncoupler or a compound that separates the process of phosphorylation from electron transport synthesis of ATP (Devlin 2010). This results in the inability of ATP being produced during the light-dependant reaction hence the inhibition of photosynthesis. 

In low light intensities the rate of photosynthesis increases linearly with increasing light intensity. The rate of increase then falls as other factors become limiting (Taylor, Green and Stout 1997).

Procedure:

MATERIALS

1. 0.35M NaCl-0.02M Tris buffer, pH 7.5 – dissolve 20.45 g NaCl, 2.42g tris (hydromethyl) aminomethane in distilled water to make 1000 mL of solution. Adjust the pH to 7.5 with 6N HCl. Store at 4˚C.

2. 0.4mM DCIP – dissolve 116mg of 2,6-dichlorophenolindophenol sodium salt in distilled water to make 1000mL of solution. Prepare just before use.

3. 0.01 N ammonia – prepare a 1N solution by bringing 6.77mL concentrated (28%) NH4OH to 100 mL with distilled water. Then bring 5.0mL of the 1 N solution to 500mL with distilled water.

4. 10-4 M DCMU – dissolve 24mg 3-(3,4-dichlorophenyl)-1,1-dimethylurea in distilled water to make 1000mL solution.

5. Purified sand.

6. Fresh spinach. Just before use, rinse with cold tap water and blot dry.

Fresh spinach leaves were collected and rinsed in cool water. The major veins were cut out and 10g of the vein free spinach leaves were weighed out. The weighed leaves were cut into small pieces and placed in a chilled mortar. Purified sand was sprinkled over the leaves. 20mL of Tris-NaCl buffer was measured and placed on ice. 15mL of the ice cold buffer was added gradually while the tissue was being ground for 3 minutes and the remaining buffer was used to rinse the mortar. 2 layers of cheesecloth were then used to filter the ground tissue into a chilled 15mL centrifuge tube. The excess juice was wrung into the tube. One drop of this suspension was taken and observed under a microscope.

The filtrate was centrifuged for 1 minute in the chilled SORVALL centrifuge and one drop of that suspension was taken and observed under a microscope. The supernatant was decanted into a clean, chilled 15mL centrifuge tube and centrifuged at 3300rpm for 5 minutes. The supernatant was decanted and the pellet kept. One drop of the supernatant was taken and observed under the microscope.

1.5mL of ice cold Tris-NaCl buffer was added to the pellet and using a Pasteur pipette, the pellet was thoroughly resuspended. The tube was covered with parafilm and inverted several times to ensure proper mixing.

This was done twice and both samples were combined into one tube. One drop of the supernatant was taken and observed under the microscope. The centrifuge tube was wrapped in foil and kept on ice and labeled chloroplast suspension.

Determination of the chlorophyll content

0.1mL of the chloroplast suspension was pipette into a small, thick rimmed test tube and 9.9mL of acetone in water was added. This was centrifuged at 2000rpm for 10 minutes. The supernatant was transferred into a glass cuvette and the absorbance read at 652nm. 80% acetone in water was used as a blank. The concentration of chloroplasts was calculated.

The volume of chloroplast suspension was measured in a 10mL measuring cylinder. Stock chloroplast suspension was diluted using cold Tris-NaCl buffer to gain a concentration of 0.4mg/mL. The total yield per gram wet weight of plant tissue was calculated.

The Effect of Inhibitors on the Rate of the Hill Reaction

Five test tubes were obtained and labeled 1-4 and the other labeled “blank”. Tube 1 was wrapped with aluminum foil and the following solutions were added in the said sequence: 3.5mL of Tris-NaCl buffer, 0.5mL of DCPIP, 0.5mL of distilled water and 0.5mL of chloroplast suspension. The tube was mixed by inversion and set aside for 10minutes. An absorbance reading was taken at the end of the 10 minutes.

The spectrophotometer was set to 600nm and the blank was prepared as followed: 3.5mL buffer, 1.0mL distilled water and 0.5mL chloroplast suspension. The spectrophotometer was zeroed.

Tube 2 was prepared using the following solutions: 3.5mL of Tris-NaCl buffer, 0.5mL of DCPIP, 0.5mL of distilled water and 0.5mL of chloroplast suspension. The absorbance was read at 600nm.

Tube 2 was then placed 25cm away from the light source. After one minute, the absorbance was measured again at 600nm and solution poured back into tube. The tube was then replaced by the light source and after one minute its absorbance read. This was done for a total of 10 minutes.

Tube 3 was prepared using: 3.5mL buffer, 0.5mL DCPIP, 0.5mL ammonia & 0.5mL chloroplast suspension. The same procedure as done in tube 2 was repeated for tube 3.

Tube 4 was prepared using: 3.5mL buffer, 0.5mL DCPIP, 0.5mL DCMU & 0.5mL chloroplast suspension. The same procedure as done in tubes 2&3 was repeated for tube 4.

The Effect of Light Intensity on the Rate of the Hill Reaction

5 test tubes were obtained and labeled 1-4 and the other labeled “blank”. The blank tube was prepared using 3.5mL buffer, 1.0mL distilled water and 0.5mL chloroplast suspension and its absorbance read at 600nm every minute for 10 minutes.

Test tube one was prepared using 3.5mL buffer, 0.5mL DCPIP, 0.5mL distilled water and 0.5mL chloroplast suspension. This tube was kept in the dark and its absorbance read every minute for 10 minutes.

Tube 2 was prepared using 3.5mL buffer, 0.5mL DCPIP, 0.5mL distilled water and 0.5mL chloroplast suspension. It was kept a distance of 10cm away from the light source and its absorbance read every minute for 10 minutes.

Tube 3 was prepared using 3.5mL buffer, 0.5mL DCPIP, 0.5mL distilled water and 0.5mL chloroplast suspension. It was kept a distance of 40cm away from the light source and its absorbance read every minute for 10 minutes.

Tube 4 was prepared using 3.5mL buffer, 0.5mL DCPIP, 0.5mL distilled water and 0.5mL chloroplast suspension. It was kept a distance of 60cm away from the light source and its absorbance read every minute for 10 minutes.

References

Devlin, Ed. Hill Reaction. 2010. http://people.hsc.edu/faculty-staff/edevlin/edsweb01/courses/Cellbiology/labmanual/new_page_4.htm (accessed October 2, 2010).

Gupta, Kamal Kumar. Cell: A Guided Tour. 2010. http://eduframe.net/andc/biology/Kamal-Bio/odlkamal.htm (accessed October 2, 2010).

Hames, David, and Nigel Hooper. 2005.Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group,

Lodish, Harvey, Arnold Berk, Lawerence Zipersky, Paul Matsudiara, David Baltimore, and James Darnell. 2007.Molecular Cell Biology. W.H. Freeman,

Nelson, David L, and Michael M Cox. 2008. Lehninger Principles of Biochemistry. United States of America: W.H. Freeman and Company,

Taylor, D.J, N.P.O Green, and G.W Stout. 1997.Biological Science 1&2 3/e. United Kingdom: Cambridge University Press,