Understanding the Hill Reaction: Photosynthesis Lab Guide
School
California State University, Long Beach**We aren't endorsed by this school
Course
BIOL 212
Subject
Biology
Date
Dec 11, 2024
Pages
9
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7-1PHOTOSYNTHESIS: THE HILL REACTION . . . . . . . . . . . . . 7 INTRODUCTION Light energy is converted to chemical energy via photosynthesis. Organisms that can photosynthesize, some bacteria, some fungi, algae, and all plants, are primary producers, or photoautotrophs. They use light energy to produce sugars, which are then utilized by the plant and eaten by herbivores. Primary producers are the basis of the food web on our planet. Organisms that benefit from producers are consumers and are considered heterotrophic. The photosynthetic reactions performed by algae, cyanobacteria and plants follow this chemical reaction: 6CO2+ 6H2O + light —> C6H12O6+ 6O2Light energy drives the conversion of CO2and H2O into six carbon sugars with the release of O2. Looking at this reaction, it is not clear whether CO2or H2O is the source of the O2that is released. In 1937 Robin Hill showed that isolated chloroplasts could release O2if a suitable electron acceptor were available such as potassium ferricyanide in which the Fe3+is converted to Fe2+. Hill noted that CO2was not consumed even though potassium ferricyanide was reduced and O2was released. The Hill reaction is a light-driven transfer of electrons from H2O to non-physiological electron acceptors against a chemical potential gradient. The Hill reaction separated O2evolution from CO2fixation, and cleanly demonstrated that H2O was the source of evolved O2since CO2was not consumed during the Hill reaction. The Hill reaction can be written as: e-acceptor + 2H2O —> e-acceptor4e-+ 4H++ O2 Robin Hill’s work showed that photosynthesis has two components -- the light reactionsevolve O2with the associated reduction of NADP+to NADPH as well as the production of ATP. These light reactions are followed by the light-independentfixation of CO2into six carbon sugars using the NADPH and the ATP formed during the light reactions. The light-independent reactions include the Calvin cycle. These light and light-independent reactions occur in different parts of the chloroplast with the light reactions occurring in the thylakoid membranes and the lumen space inside of the thylakoids and the light-independent reactions occurring in the stroma. Figure 7.1 provides an excellent overview of the light and light-independent reactions. Today, you will be isolating thylakoid membranes from spinach leaves using a simple differential centrifugation protocol (simple version of the technique used during cellular fractionation of cauliflower). Thylakoids contain proteins that bind chlorophyll and other pigments, so the thylakoid membranes will be a deep green color. It is important that all steps be done on iceso that the biochemical activity is retained in the thylakoid membranes. Leaf tissue will be ground up in a blender to break cell walls and then filtered to remove debris. The filtrate will be spun at low speed to remove nuclei and cell wall fragments, and the supernatant will be saved. This supernatant will then be spun once more, at greater x g, and the thylakoid membranes will sediment to the bottom of the tube. The green pellet will then be resuspended, and the thylakoid
7-2suspension will be used in today’s lab, the Hill reaction. Although Robin Hill used potassium ferricyanide as the electron acceptor, you will use DCPIP, the same electron acceptor that was used in the succinate dehydrogenase assay. Recall that oxidized DCPIP is blue and that reduced DCPIP is colorless. As photosynthesis occurs, O2will be released (bubbles will form in reaction tube) and the blue color will decrease as DCPIP is reduced. As before, progress of DCPIP reduction will be followed by a decrease in optical density at 600 nm. We will use DCPIP’s Molar extinction coefficient of 0.016 Molar-1cm-1to estimate the rate of the light reactions of photosynthesis. For this lab, you will determine whether the reduction of DCPIP in the Hill reaction requires light and whether the reaction rate is faster with higher or lower intensity light. What do you expect the answers to be? Can you relate your answers to the weather that outdoor plants encounter?Figure 7.1. A chloroplast showing the reactants, products, and locations of the Light reactions and Light-independent (Calvin Cycle). PROCEDURE A. ISOLATION OF THYLAKOID MEMBRANES Note before you begin:it is critical that all collected fractions be maintained on ice and that all materials to be used be chilled to maintain enzyme activity. Be sure that you understand the experiment thoroughly before you begin. 1.Instructor: Transfer 35 g of fresh spinach and 100 ml of ice cold 0.4 M NaCl to the pre-chilled glass blender jar. a.Grind the tissue for 1 minute and then filter the resulting homogenate through 8 layers of cheesecloth into a pre-chilled 250 ml beaker. b.Maintain the beaker on ice.
7-3_______________________________________________________________________ 2.Students: Label two 15 mL tubes with the name of your partnership. Place these tubes on ice to cool. 3.Transfer 4 mL of the homogenate into 1 of your chilled 15 mL tubes. Centrifuge this tube at 200 x g (1500 RPM) for 3 minutes at 4ºC. a.Make sure to balance your tube with that of another group. 4.Using our P1000, carefully transfer the supernatant from the tube into a new prechilled conical tube. a.The pellet consists of unbroken cells and cell wall debris. It will be small and difficult to see until the supernatant is nearly all transferred. b.Following the transfer, the tube containing the pellet can be discarded. 5.Centrifuge the tube containing the transferred supernatant at 2200 x g for 10 min at 40C. a.Be sure to balance your tube with another partnerships. 6.Using your P1000, carefully remove and discard the supernatants from the tube. Once the supernatant has been removed, transfer 2.0 mL of ice cold 0.4 M NaCl to the tube. 7.Resuspend the pellet by gently pipetting up and down numerous times. Once the pellets have been fully resuspended, centrifuge at 2200 x g (5000 RPM) for 10 minutes at 4ºC. a.Be sure to balance your tube with another partnerships. 8.Using your P1000, carefully remove and discard the supernatants from the tube. Once the supernatant has been removed, transfer 2.0 ml of ice cold of 0.05 M phosphate buffer solution to the tube. 9.Resuspend each pellet by gently pipetting up and down numerous times, making as uniform a suspension of thylakoids as you can. This is your suspension of active thylakoid membranes. a.KEEP AT 40C UNTIL NEEDED B. REDUCING POWER AT VARIOUS LIGHT INTENSITIES 1.Label 3 glass spectrophotometer tubes (1, 2, 3) and pipette into each tube put: a.2.0 mL of 0.05 M phosphate buffer b.500 μL of the 0.8 mM DCPIP (blue indicator dye). c.Into tube 3 ONLY, pipette 15 μL of 5% (w/v) ascorbic acid. i.Tube 3 will serve as a spectrophotometer blank during the experiment.
7-4ii.Ascorbic acid is an electron donor (reducing agent). Note that the ascorbic acid immediately reduces the DCPIP indicator dye, and the blue coloration is lost. 2.Make aluminum foil sleeves for tubes 1 and 2. These will keep light from entering the tubes. For all tubes, prepare a foil cap and a label it. 3.Prepare an ice water bath by filling a 250 mL beaker approximately half full with ice. 4.Place tubes 1 and 2 in their foil covers. Pipette 500 μL of the active thylakoid membrane suspension into each of the three tubes. a.Attach the foil cap, hold it closed tightly and invert each tube three times. Be sure to hold the cap firmly on the mouth of each tube with your thumb to prevent any leakage while mixing. 5.Place all three tubes in the ice water bath. Your group will be assigned either a high lightor low lightintensity treatment. If you are at the high lighttreatment, place the beaker 15 cm away from the lamp bulb. If your group is at low light, place the beaker 30 cm away from the lamp bulb. a.Be sure tubes 1 and 2 are covered with their aluminum foil sleeves. Always keep all tubes on ice except when a particular tube’s absorbance is being read. Please see figure 7.2 for set-up. Figure 7.2. Be sure to place tubes 1 and 2 (with foil caps and sleeves) in ice water 15 (high light) or 30 cm (low light) from the light source. Tube 1 will be the light exposure treatment and tube 2 will be the dark control. You will need to obtain data from the other partnership that performed the Hill reaction at the other light intensity.
7-56.Set the spectrophotometer to 600 nm and zero the absorbance using your blank - Tube 3. The lights in the laboratory will be switched off at this point to conduct the experiment. a.NOTE: Invert to mix and wipe dry the tubes with a Kimwipe before placing them into the spectrophotometer to take an absorbance reading.7.Remove the foil covers from tube 1 (the experimental tube) and 2 (the dark control tube) and take their absorbance readings. Record these readings for 0 minutes of light exposure in Table 7.1. 8.After reading the tubes, replace the foil cover on tube 2 ONLYand place all tubes back in the ice water bath such they are against the side of the beaker facing the lamp. 9.Switch on the lamp. At the end of 2 minutes, obtain an absorbance reading for tube 1. a.Always dry the spec tube with a Kimwipe before taking your reading. 10.Repeat this process of light exposure in 2-minute intervals for a total of 14 minutes. Remember to switch the light off after each time interval while taking absorbance readings. Record all data in Table 7.1. 11.At several times during the experiment (at 0, 6 and 12 minutes), quickly remove tube 2 (the control) from its foil sleeve and read its absorbance. Immediatelyreturn this tube to its foil sleeve and place it back in the ice water bath. Its absorbance should remain constant provided it has been maintained in the dark. Record the data in the Table 7.1. TABLE 7.1Absorbance Time (minutes) Experimental*- Low Light Intensity (Tube 1) Experimental*- High Light Intensity (Tube 1) Control- Dark Tube (Tube 2) 0 2 4 6 8 10 12 14 *One set of data will come from another group using a different light intensity. Graph & Calculations (12-15): Graph & calculations for both low and high light intensities. 12.Graph the absorbance of tubes 1 and 2 (y-axis) verses time (x-axis).
7-6a.Include the data for tube1 from another group that did a light intensity different from your partnerships. b.Your graph should have 3 lines: low light intensity, high light intensity and dark (control). c.Draw best fit line through the initial points for each treatment. d.Using each line, extrapolate to the x-axis to determine the length of time required for complete reduction of the DCPIP for both light intensities. 13.Using the extinction coefficient of 0.016 Molar-1cm-1for oxidized DCPIP, calculate the rate of reduction of DCPIP (Mmin-1) from your change in absorbance over time and the extinction coefficient. a.Take the absolute value of the slope to find the change in absorbance/min. b.absorbance/min = e in Molar-1cm-1* C * 1 and solve for c in Molar/min 14.The typical yield of thylakoids is equivalent to 5 x 107chloroplasts per mL. Using this concentration of chloroplasts and a reaction volume of 3.0 mL, calculate the rate of reduction of DCPIP per chloroplast per minute in moles x chloroplasts-1 x min-1 for both light intensities used. a.Molar/min from 13.B convert to moles/min. Then using the reaction volume (3 mL) & the conversion of Molar to moles. b.1 mole of DCPIP accepts 2 moles of electrons. 15.Recall that only 0.5 ml of thylakoid suspension was used for each tube. If one molecule of DCPIP requires two electrons to be reduced, calculate the number of electrons transferred per chloroplast per minute for both light intensities used. a.The number of electrons in 1 mole is Avogadro’s number(6.023x1023). b.You now have electrons/min, divide by # of chloroplasts used (0.5 mL of 5*107chloroplasts/mL) c.Final positive numbers in #electrons transferred per chloroplast per min. Questions
7-71) Draw two molecules of H2O each with eight electrons. Show the O2(12 electrons) as well as the four electrons and four protons that are produced during the Hill reaction. 2) A) Why did the Hill reaction prove that CO2was not the source of O2that is released during photosynthesis? B) Which molecule is the source of O2? 3) What products of the light reaction are used to fuel the light-independent reactions which fix CO2into six carbon sugars? 4) A) Why did the A600decrease as the Hill reaction took place? B) Could the products of the Hill reaction be used to drive the light-independent reactions? C) What do the four electrons reduce during the light reactions when there is no artificial electron acceptor present?
7-8
7-9Portions of the protocols used in this lab were adapted from the following source: Mason, A. Z., Carlberg, D., Gharakhanian, E., Brusslan, J., and Palmier, C. 2004. Laboratory Manual For Biological Sciences I, 7thed. Pearson Custom Publishing.