Photosynthesis is the process by which phototrophs use water, carbon dioxide, chloroplasts, and light energy to make their own food. There are two smaller reactions that make up photosynthesis. These are the light independent reaction, and the light dependent reaction, or the Hill reaction. The reaction for photosynthesis is H¬2O + CO2 + light yields (CH20)N +O2. The Hill reaction is the light dependent part of photosynthesis, and occurs in the chloroplasts of the cell. Using light energy, the chloroplasts oxidize water and use the elections to reduce NADP+ to NADPH and to synthesize ATP. In this reaction, the blue dye, DPIP, can be used to replace NADP+ in its role as an electron acceptor.
When DPIP accepts electrons, it becomes translucent, a property that can used to measure the rate of photosynthesis. This amount of light that can pass through DPIP that has been reduced is called transmittance and can be measured by a spectrophotometer. This can be used to test the rate of photosynthesis. Since it is known that as DPIP is reduced it become translucent, and that heat denatures proteins, it can be hypothesized that if a solution of chloroplasts is heated up, the rate of photosynthesis will drop and the transmittance of the solution will also drop (Stegenga).
To test the hypothesis a solution was made from a buffer, water, DPIP, and a chloroplast solution. One mL of a buffer was added to maintain a constant pH. Three mL of water was added to the sample tubes to provide water for the Hill reaction. After those were added to a tube, one mL of DPIP was added to the sample tubes to replace the NADP+. It is important that the DPIP is added before the chloroplasts because if the chloroplasts are added first the Hill reaction will start without the DPIP, using NADP+ as it normally would. The chloroplast solution was made from spinach leaves. The stems were removed as they did not have the organelles of interest, and the rest of the leaves are left under a light to start photosynthesis reaction in leaves. The leaves are added to .5M sucrose. Sucrose is used instead of water because sucrose is isotonic to the leaves. The solution is blended for three ten second bursts. It is blended in multiple bursts to reduce the amount of heat in the solution so that the proteins inside do not denature. Then the solution was poured through a cheesecloth to filter out unwanted organelles.
Three drops of the chloroplasts was added to each tube. Each five mL solution would then be put in the spectrophotometer at 0, 5, 10, and 15 minutes after being made. While the solution was waiting it was under a lamp to ensure photosynthesis. A beaker of water was placed between the lamp and the beakers to reduce heat. Each tube is covered with parafilm and wiped to reduce outside interference. It is wiped to reduce fingerprints that will affect the transmittance. For this experiment there will be four tubes. A calibration tube filled with 1 mL of buffer and 4 mL of water, and three drops of chloroplast totaling 5 mL of fluid. No DPIP was added so that the transmittance never changed. The three sample tubes were made of 1 mL of buffer, 3 mL of water, one mL of DPIP, and three drops of chloroplasts.
There was two water baths set up. One at room temperature and one at a heated temperature. There was one tube in the room temperature as a control. It was placed in its own water bath so it gets a similar amount of light. Then two were placed in a heated water bath. One was covered completely with aluminum foil to prevent photosynthesis. This is the second control. The third tube will be identical to tube one, but placed in a heated water bath. The rate of transmittance is measured as the change in transmittance divided by the change in time. The transmittance was used because the rate of change is measured by the transmittance. As photosynthesis occurs, the DPIP will become transparent, so the higher the transmittance, the higher the rate of photosynthesis (Stegenga).
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