Purpose
One purpose of this lab is to test the effect of light intensity on photosynthetic activity.
Another purpose of this lab is to test the effect of boiled versus unboiled chloroplasts on photosynthetic activity. Variables
1: Independent
The independent variable is the light intensity (presence or absence of light).
Dependent
The dependent variable is the percent transmittance (%). This will be measured by a colorimeter.
Controls
Same Volume of total liquid, Volume of Phosphate buffer, Volume of chloroplasts
2: Independent
The independent variable is the amount of heat the chloroplasts were subjected to (boiled versus unboiled).
Dependent
The dependent variable is the percent transmittance (%). This will be measured by a colorimeter.
Controls
Same Volume of total liquid, Volume of Phosphate buffer, Volume of chloroplasts
Hypothesis
As the light intensity decreases the percent transmittance will not increase, it will remain constant. Photosynthesis is the process of taking an inorganic form of carbon and transforming it into a storable energy rich organic sugar. In many plants this is the basic energy that fuels many of the processes. This reaction of photosynthesis, however, cannot work without light. The first reaction phase of photosynthesis is called the light dependent phase. In this phase water molecules are broken up to supply electrons to photosystem 2. When light strikes antenna pigment molecules energy is transferred to these electrons. The energy moves through photosystem 1 where the difference in voltage created by the movement of the electron is used to produce ATP. These electrons are then accepted by NADP where they are transported to the Calvin-Benson cycle. If there is no light, there is no way to break up the water molecule and create electrons that would eventually oxidize the NADP (DPIP in this case). Thus without DPIP being used the transmittance will not decrease either.
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As the amount of heat the chloroplasts are subjected to increases (boiled) the percent transmittance will not increase or it will stay relatively constant. When proteins like the chloroplasts are subjected to such large amounts of heat, akin to boiling the chloroplasts, they begin to lose their quaternary, tertiary and secondary structures. Even the smallest change in conformation of a protein will cause the protein to lose its function. In this case the reactions necessary to absorb the light and breakdown H2O (by means of Manganese cubane clusters*) will be lost. Thus, without being able to perform these functions, the chloroplasts will not produce or move any electrons through the thylakoid membrane to eventually oxidize the NADP (DPIP in this case). Thus without DPIP being used the transmittance will not decrease either.
*PERSPECTIVES BIOCHEMISTRY: Water Photolysis in Biology A. W. Rutherford and A. Boussac (19 March 2004) Science 303 (5665), 1782. [DOI: 10.1126/science.1096767]
Procedure
First calibrate the colorimeter with blank cuvette number 1. The instructions on creating cuvette one is listed in the Procedure table. Next prepare cuvettes 2-5 by following the instructions listed under the procedure table. Make sure that each of the cuvettes stays in ice to keep the chloroplasts in ideal conditions. Also make sure that cuvette number two stays wrapped in aluminum foil so that no light get through. The aluminum foil must be wrapped so that it is easy to take out the cuvette and replace it. Now start by putting cuvette number two into the colorimeter and record the percent transmittance after it has leveled off. Now start the timer and record the starting time. Follow the same procedure for the other 3 cuvettes, taking their initial percent transmittance. Place each one in front of the heat sink after the transmittance has been read. After 5 minutes has past after each initial reading, take a second reading and then a third and fourth reading.
The hypothesis was proven correct by the experiment. As the light intensity was increased in the experiment the Percent transmittance also increased. In the trail with the dark condition (cuvette 2), the transmittance went from 17.5% to 19.32% 19.9% finally to 18.9%. This fluctuation was probably due to the experimental error that was a result of a messy procedure. Every time that the cuvette was taken out of aluminum foil wrapper and placed into the colorimeter it received some light that it should not have received. In this case the one that did receive light saw a major jump from 18.088% transmittance to 84.631% transmittance in 5 minutes. This result was seen because the light provided the energy to break up H2O molecules and energize electrons. These electrons went on to reduce the DPIP molecules which turned clear after the reaction. This color change allowed us to read the progression of the photosynthesis and the rate at which it was occurring. In this case we saw that very quickly the DPIP was reduced and thus changed from colored to clear, thus increasing the percent transmittance. The reason that the dark (light absent) cuvette had little fluctuation was due to the fact that there was no energy supplied to break up the H2O and provide electrons for the reduction of DPIP. Thus there was no change in transmittance
The hypothesis was proven correct by the experiment. As the chloroplasts were subjected to more and more heat (boiled) they began to denature and they were not able to conduct the reactions necessary to sustain photosynthetic activities. This denaturing occurs due to the breaking of the bonds that hold the protein together. These bonds include: the quaternary structure which involves the specific shape formed while in chaperone molecules, the tertiary structure includes interactions between side groups of amino acids and the secondary structure which involves hydrogen bonds between amino acids. As these levels of protein structure begin to break down, receptors lose their shape and cannot carry signals. In this case the chloroplast’s key enzymes and proteins lose their form and thus cannot perform oxidative phosphorylation necessary to produce ATP for the Calvin cycle. As the temperature increases a number of other functions are also left unfulfilled as the chloroplasts begin to lose their shape and purpose. The data that was taken from this experiment shows this same trend. In Cuvette 4 which contained the boiled chloroplasts, the transmittance went from 24.3% to 27.5% to28.5% to finally 30.1% transmittance. This shows a relatively small change and while considering that DPIP is light sensitive these results are acceptable. In cuvette 3 which contained unboiled chloroplasts a major jump was from 18.088% transmittance to 84.631% transmittance in 5 minutes.
Analysis
Part A
The solubility of the pigments in the solvent affects the separation of the pigments. The pigments are carried different distances because they are not all equally soluble. And because the solvent carries these pigments up the paper the less soluble pigments get left closer to the base while the most soluble ones go farther along the paper. There is also a separation due to the different attractions of the pigments to the fiber of the paper.
No I would not expect the Rf to be the same because with a new solvent the solubility of each of pigments has now changed. Also when a new solvent is introduced the amount that the solvent will move up the paper will change. Since Rf is calculated by dividing the distance traveled by the pigment by the distance traveled by the solvent, thus with both numbers changing the Rf will change.
The reaction center contains both chlorophyll a, carotenes, xanthophylls, and chlorophyll b. These pigments work together to capture light energy and help transfer them to electrons. These new higher energy leveled electrons now help conduct oxidative phosphorylation. Another function of these pigments is to protect the plant from UV light.
Part B
The DPIP in this experiment acts as a colored substitute for NADP which normally accepts electrons at the end of the light dependent reactions of photosynthesis. It is important that it is reduced by the free electrons so we can measure the rate of photosynthesis. It is also important that it is a colored compound so that we are able to use colorimetry to measure the rate of the disappearance of the DPIP as it turns clear as it is reduced.
DPIP replaces NADP.
The electrons come from the hydrolysis of water.
The spectrometer measured the percent transmittance in this experiment. This is an important measure for it shows that the DPIP is accepting electrons as it turns clear. The higher the transmittance the more DPIP has accepted an electron. The spectrometer itself measures the amount of light that is transmitted or absorbed through a cuvette.
Without light electrons do not attain a higher energy state and move across the thylakoid membrane and to the DPIP. Without electrons the DPIP is not reduced and does not turn from colored to clear.Boiling the Chloroplasts denatures the proteins and thus the chloroplast’s key enzymes and proteins breakdown. These enzymes are key in the absorption of light and the excitation of electrons for movement through the thylakoid membrane. If the electrons don’t move out they cannot reduce the DPIP.
The chloroplasts kept in the dark do not receive any light. Without light, electrons cannot be excited and thus they do not along the thylakoid membrane where they would eventually be accepted by DPIP. The DPIP will not be reduced and will not turn clear as a result.
his will cause the transmittance to stay the same. The light chloroplasts on the other hand will move the electrons when they are excited and the electrons will reduce the DPIP. This will cause a color change from colored to clear causing the transmittance to increase.
Cuvette 1
– Serves to calibrate the spectrometer for use in the experiment.
Cuvette 2
– A cuvette used to show the effect of an absence of light on photosynthetic activites.
Cuvette 3
– Used to show the effects of light on photosynthetic activities. While both cuvette 2 and 3 both have unboiled chloroplasts, cuvette 3 also serves to test the effect of unboiled chloroplasts on photosynthetic activity.
Cuvette 4
– Used to show the effect of boiled chloroplasts on photosynthetic activities.
Cuvette 5
– Functions to show that it is the chloroplasts that are causing the DPIP to break down. Acts as the control.
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