LAB 5: The HILL REACTION
Bio207 – Cell Biology
Th photosynthetic organisms can attract light energy and use it in sugar formation and oxygen productions, which was a fantastic ability. The primary purpose of this experiment to determine the rate of photosynthesis in different light colors grounded spinach leaves. In most cases, the declining capacity for the evolving oxygen was temporarily color-sensitive during the process of isolated chloroplast disintegration. During the process of photosynthesis, a natural acceptor NADP was reduced to NADPH in the chloroplast. Furthermore, it was also used to assess the effect of light intensity at the rate of hill reaction and determine the impact of inhibitors on the response of the hill reaction using DCPIP as an artificial reaction acceptor and spectrophotometry absorbance 600mm.
Photosynthesis is when solar energy is trapped and used for the carbohydrate synthesis from Carbone dioxide. In most cases, the physiological process occurs in green plants, photosynthetic bacteria, and algae. The physiological process is facilitated by the inhibitors called photosynthetic organisms capable of capturing light energy and using it from sugars and oxygen gas. Therefore, it a fantastic ability. Therefore, to conduct this experiment, spinach was the preferable specimen to be used since they are easy to come by. They can perform photosynthetic experiments in any environment. The process of photosynthesis underwent two processes such as photosystem one and photosystem two, and in which experiment had also had two parts (Pridham, 2008). The chloroplast organelle had also been isolated from the spinach leaves.
Additionally, the chloroplast could have also been isolated from the rest of the cell components through centrifugation, which was based on the weight of the chloroplast. According to Blackman, he suggested that photosynthesis processes are composed of two major series of reactions. The first part of the reaction is called a light reaction or light-independent reaction. These types of reactions are majorly depending on energy and trap the radiant energy. On the other hand, the dark independent stage does not require light but uses energy captured during the light stage for the organic molecules’ synthesis. Robert Hill did the first chloroplast isolation in the presence of light. After isolation, the ability of the chloroplast to oxidize water which is the first step of photosynthesis. Which is performed by the Hill Reactions (Pridham, 2008). During the light-independent stage or reaction of photosynthesis, water in the plant is oxidized to provide electrons for the chlorophyll pigment. The main reason why Hill’s experiment just involved the light reaction stage is that if he wanted to prove that it uses compounds that use the energy of light. To determine the impact and effectiveness of the reaction, he used an equation to determine the process.
H2O + DCIP (oxidized)Blue color + DCIP (reduced) colorless + ½ O2
A change in absorbance is used to measure the rate of the Hill Reaction or the photosynthetic. DCIP was reduced and became colorless when chloroplast is subjected to light energy. At the same time, DCIP will increase in the concentration of 4x 10-4m which its molecular weight is 290.08g/mole. Experiments will be conducted in two parts, that is Chloroplast Isolation and Hill Reaction using Isolated Chloroplast.
The main reagents and the specimen used include DCIP and Ammonia. An ice bath and pre-cool all glassware and motor and pestle were prepared, and fresh spinach leaves were selected. The large veins that were found in the center of each leaf were removed. 6.0 grams of deveined leaf tissue were weighed then chopped using a scalpel. 15 ml of ice-cold Tris NaCl was added, which was made earlier this semester, to a motor, and then the chopped spinach was added. The mixture was ground into a fine paste.
The paste was left for three minutes. A piece of cheese clothes about 10 cm wide was cut and then folded into two halves. The pieces were then placed over the beaker, and the solution was filtered by squeezing the tissue pulp to recover most of the liquid. 1 mL was then transferred from that liquid into 6 Eppendorf tubes aliquot proportion of the given material—centrifuge at 200xg (at speed for 2) for approximately 1 minute. The Eppendorf was placed in the centrifuge with half on one side and the other half on the other side. The Eppendorf was hinged on the outside. The cap was kept on the rotor, and the tops were also closed. The speed of the centrifuge was checked, which is set to 2. Then the quick spin was held for approximately one minute.
The pellet was then unbroken cell material and plant cell walls. The supernatant liquid was pipetted into a new Eppendorf tube, then thrown away with the pellets in the container waste. Centrifuge at 10000xg (set speed for 10) for seven minutes. Change the speed dial of the centrifuge to 10—Balance the Eppendorf in the centrifuge. Turn up the timer to 7 minutes. The pallet is the chloroplast. Remove the supernatant with a pipette. The liquid is waste, and the pallets are the chloroplasts. The speed dial of the centrifuge was changed to 10, and the Eppendorf was in the centrifuge was also balance. The timer was turned up for 7 minutes. The pallet was used as a chloroplast. 0.75 mL of the ice-cold Tris-NaCl buffer was added to every pallet and resuspended by pipetting up and down until it got dissolved in the buffer. The materials from all 6 Eppendorf tubes were put together into one glass test tube. Furthermore, the glass tube that contained chloroplast was isolated from the spinach leaves. It was then covered and kept in the beaker of ice, a place it cannot be knocked over.
The four cuvettes were suspended in a cuvette holder and labeled near the top with B, 1,2,3.
Using this table add reagents to each cuvette.
|Cuvette||Buffer||DCIP (blue)||Distilled water||Chloroplasts|
|B(blank)||1.75 mL||0 mL||500 ul||250 ul|
|1||1.75 mL||250 ul||250 ul||0 mL|
|2||1.75 mL||250 ul||250 ul||0 mL|
|3||1.75 mL||250 ul||250 ul||0mL|
The computer was turned on, and plug in the Spectro vis to the USB port. Open LoggerPro program. To set up the spectra Vis for the Hill Reaction. A. click on the data collection button. That is the button on the top right. Use the menu to set the type ” Time Based.” Set Duration for 60 seconds Set sampling rate to 01, samples/ second 10 seconds/ per samples. THEN THE DONE BUTTON WAS CLICKED. Click on the configure spectrophotometer Button. The material was then checked whether it got mixed up. Place cuvette into Spectra Vis with stripes front and back. In the experiment, the menu selects calibrate the select Spectro Vis. In the menu window, allow Spectro to warm up, then select “Finish Calibration.”. When calibration is complete, then click OK. Bright Light Hill Reaction.
Take cuvette#1 and wipe with Kimwipe. Cuvette was placed in spectra Vis. A lamp nearby turned on so that it is ready. Put v250 ul of chloroplast in your P 100 micropipette tip. DO NOT ADD IT YE. AT THE SAME TIME, DO THE FOLLOWING STEPS. Add. 250 ul. of chloroplast solution to cuvette in Spectro Vis. Press the Triangle button to start recording data. Place white light on top of Spectro vis WAIT FOR 60 SECONDS! after completing your essay, then. Record your data in a table on the next page in the experiment menu, select “Store Latest Run” THIS STEP IS NECESSARY. Medium Light Hill Reaction. Step 4 above was repeated with cuvette #2. The only change was to place a plastic weigh boat on top of the cuvette during assay to block some lights like clouds. Low light Hill reaction step 4 above was repeated with cuvette #3. the only change is not using a lamp and cover cuvette to block all light from the room.
The provided data illustrate the reducing power characteristics of the chloroplast by taking absorbance reading 600 nm at the intervals of light exposure intervals of 10 seconds. The procedure was repeated for each bulb at their light intensity measurement of 250 u moles m-2 sec-1 and higher.
|Absorbance at 600nm|
The data were collected through observation and note-taking. The red color had a high absorbance rate ( 1.114), followed by green and then white.
Considering the absorbance rate at 600nm, the red bulb had a high absorbance rate, green and white. Additionally, the high absorbance rate was detected the zero seconds and at tenth seconds. However, the absorbance rate decreases with an increase in time. The interval time for table 1 is 10 seconds. Lastly, the green bulb recorded the highest absorbance rate while the white bulb recorded the lowest,
Table 2: Rate of Photosynthesis Using Colored LED Bulbs
|Change in Absorbance at 600nm|
Experiment B was conducted when the absorbance rate was varying. Zero results were recorded at zero minutes. White recorded the highest record, followed by red color and green color. The data from this table was collected by observation and note-taking. Marjory, the data from this experiment did was not above 1.0. Additionally, the data ranges from 0.0 to 0. 372. Furthermore, there is a downward projection since the value of each color increases with an increase in time.
The plots demonstrate absorbance rate 600 nm vs. time (seconds) for each bulb at their light intensity measurement of 250 u moles m-2 sec-1 and using the data taken from the data from table B.
As it is initially hypothesized our result, we proved that light-independent reaction of the photosynthesis requires photon of light as energy, however, for the second part. B, the results were expected that the absorbance light would have decreased as the distance increase. We got the results since we used CDIPIP as our substitute for NADP+ for us to show to indicate how it accepts energy electrons captured from sunlight, bulbs in our case (Figg et al. 2018). When we considered the absorbance rate for the bulbs decreased with time, each bulb increased at their colors. However, sometimes an error might occur between our absorbance readings because we noticed that the white bulb has the lowest absorbance measurement when it should be green since it has the highest intensity of 250 m-2 sec-1. Sometimes, the error could have come from spectrophotometer readings for our absorbance. However, even though an error occurred, it did not affect the results (Figg et al., 2018). In conclusion, after testing the hypothesized result in the compound formation that has light energy and therefore was able to determine that the light intensity increase, the reduction of DCPIP increases to the point where the light photon exists chlorophylls cells that saturated the energy.
Bio207- Cell Biology – Lab Manual, 2012.
Figg, C. A., Hickman, J. D., Scheutz, G. M., Shanmugam, S., Carmean, R. N., Tucker, B. S., … & Sumerlin, B. S. (2018). Color-Coding Visible Light Polymerizations to Elucidate the Activation of Trithiocarbonates Using Eosin Y. Macromolecules, 51(4), 1370-1376.
Mai, R., Symmank, C., & Seeberg-Elverfeldt, B. (2016). Light and pale colors in food packaging: When does this package cue signal superior healthiness or inferior tastiness? Journal of Retailing, 92(4), 426-444.
Pridham, T. G. (2008) Color and Streptomycetes: report of an international workshop on the determination of the color of Streptomycetes. Applied Microbiology, 13(1), 43-61.
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