Lab 7: Photosynthesis (Revised Fall 2008) Adapted from Expmt 7 in Biology with Computers Lab 7 - Biol 201- Page 1 of 12 Lab 7. Use of DPIP Color Changes to Monitor the Rate of Photosynthesis Prelab Assignment Before coming to lab, read carefully the introduction and the procedures for each part of the experiment, then answer the prelab questions at the end of this lab handout. Hand in the prelab assignment just before the start of your scheduled lab period. Goals of this Lab After completing this lab exercise you should be able to..... - Use a computer and a colorimeter to measure color changes due to photosynthesis. - Explain the effect of light on the rate of photosynthesis. - Explain the effect that the boiling of plant cells has on the rate of photosynthesis. - Compare and explain the rate of photosynthesis for a chlorophyll extract with a chloroplast extract. - Compare and explain the rates of photosynthesis for plants in different light conditions. Introduction The process of photosynthesis involves the use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds. 6 H2O + 6 CO2 + light energy C6H12O6 + 6 O2 Equation 1. The net equation summarizing the overall process of photosynthesis leading to the formation of glucose. This process is an extremely complex one, occurring in two stages. The first stage, called the light dependent reactions (or the “light reactions”) of photosynthesis, requires light energy. The products of the light dependent reactions are then used to reduce carbon dioxide to produce sugars such as glucose. Because the reactions in the second stage do not require the direct use of light energy, they are called the light independent reactions or the Calvin Cycle. In the light dependent reactions, electrons derived from water are “excited” (raised to higher energy levels) in several steps, involving photosystems I and II. Light energy is absorbed by Chlorophyll molecules in each photosystem to excite electrons. Normally, these electrons are passed to a cytochrome containing electron transport chain. In photosystem II, these electrons are used to generate ATP. In photosystem I, excited electrons are used to produce the reduced coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). Both ATP and NADPH are then used in the Calvin cycle to produce glucose. ATP is used as an energy source to drive the endothermic Calvin cylce, while NADPH is used as a source of hydrogen atoms and energy to reduce carbon dioxide to form sugars such as glucose. Note: During the light reactions, the processes involving photosystem II occur before those involving photosystem I. The numbers are reversed simply because photosystem I was discovered before photosystem II.Lab 7: Photosynthesis (Revised Fall 2008) Adapted from Expmt 7 in Biology with Computers Lab 7 - Biol 201- Page 2 of 12 2NADP+ (oxidized) + H2O + light energy  2 NADPH(Reduced) + 2 H+ + O2 Equation 2. The light dependent reactions use light energy to reduce NADP+ to form NADPH by adding a pair of electrons and a hydrogen ion that were generated by the spliting of a water molecule. ATP, not shown in the equation above, is also produced in the process. Oxygen is released as a by-product. In this experiment, a blue dye (2,6-dichlorophenol-indophenol, or DPIP) will be used to replace NADP+ in the light reactions. When the dye is oxidized, it is blue. When reduced, however, it turns colorless. Since DPIP replaces NADP+ in the light reactions, it will turn from blue to colorless when reduced during photosynthesis. This color change will allow you to monitor the rate of photosynthesis. DPIP (oxidized) + Water + light energy DPIP(Reduced) + Oxygen (blue) (colorless) Equation 3. The color change of DPIP from blue to colorless will be used to monitor the rate of photosynthesis In order to allow the DPIP to come into contact with chloroplasts, the plant cells will need to be carefully disrupted to release the chloroplasts into the buffer solution. Placing spinach leaves along with a buffer solution in a blender to gently break open the cells easily produces a chloroplast suspension. This suspension will then be used to test for photosynthetic activity. The intensity of color, measured as absorbance, will be detected by a computer-interfaced colorimeter. The amount of red light (wavelength,  = 635 nm) absorbed by the DPIP - chloroplast suspension indicates the amount of oxidized DPIP present. According to Beer’s Law, the concentration of solute (oxidized DPIP in this case) is proportional to the amount of light absorbed by that solute. The more red light absorbed, the more oxidized DPIP present, and vice versa. Beers Law: Absorbance  Solute Concentration But why use red light to determine the relative amount of oxidized DPIP present? DPIP in the oxidized state has a blue color because oxidized DPIP molecules absorb almost all of the wavelengths of visible light except the blue wavelengths. Hence, the blue wavelengths are transmitted and reflected from the solution, thus producing its blue color. Since DPIP in the oxidized state absorbs wavelengths in the red end of the spectrum, the amount of red light (e.g. = 635 nm) absorbed will, according to Beer’s Law, be an indication of the amount of oxidized DPIP present. But as photosynthesis occurs DPIP becomes reduced and the blue color of the solution starts to fade as more and more of the colorless reduced form is generated. Figure 1. The Vernier colorimeter measures the percentage of light of a specific wavelength that passes through the cuvette (i.e. The % Transmittance is measured). Absorbance, the amount of light absorbed by the cuvette and its contents, is then calculated from the %T by the LoggerPro software by using the relationship: Absorbance = - log (% T / 100).Lab 7: Photosynthesis (Revised Fall 2008) Adapted from Expmt 7 in Biology with Computers Lab 7 - Biol 201- Page 3 of 12 MATERIALS Per Group of Two Students: Per Class: Goggles GoLink Vernier Colorimeter 2-Cuvettes with lids 1-Cuvette covered with foil with lid One 5-mL pipette Pipette pump or bulb 2-Beral pipettes 250 mL beaker (filled with ice) Two small test tubes 600 or 1000 mL