Isolation of Chloroplasts


Introduction:

The purpose of the experiment is to isolate chloroplasts from plant and to investigate the function of them.
In nature, three kinds of plastids exist and one of three is chloroplast. The root of this organ where photosynthesis takes place is based on endosymbiotic bacteria and each leave of plants have too much chloroplasts. Basically, photosynthesis, producing ATP and NADPH by using light and water for generating glucose, can be occurred by all green plants.(1)
The structure of chloroplasts is formed some basics. These are outer and inner membranes, intermembrane space, stroma, thylakoid system. These two-bounded organelle have an outer membrane that small molecules can be diffuse through membrane, but also protein have a large structure cannot penetrate the membrane. Stroma is surrounded by inner membrane that has transmembrane proteins on it. It regulates passage of molecules. Stroma is semi fluid and contains chloroplast DNA, ribosomes, thylakoid system, starch and many proteins. Thylakoid system is made of membranous sacks known as thylakoids. Chlorophyll pigments are in them and because of that, light reactions of photosynthesis happen there.(2)
Photosynthesis has two stages to produce organic molecules, light-dependent and light-independent reactions. At light-dependent reactions, important protein complexes being in the membrane of thylakoids are assigned. These are Photosystem Ⅰ and Photosystem Ⅱ. They absorb light and use energy for transporting electrons. Also this electron transport system is known as Electron Transport Chain (ETS). The passage of these electrons is used for pumping H+ ions into thylakoid space or lumen. This H+ source prides from water by using light. H+ ions form a concentration gradient between stroma and thylakoid lumen. ATP synthase produces ATP while H+ ions passing. Besides while electrons are transport from a molecule to another molecules, NADPHs are produced. (2)
At light-independent reactions or dark reactions, CO2 taken from atmosphere is used for producing carbohydrates by being modified. Also this process is called as carbon fixation. CO2 is combined with a 5-C sugar, ribulose 1,5-biphosphate. With reactions, unstable molecules are converted to glucose by using ATP and NADPH produced at light-dependent reactions. (2)
Method and Material:
Part 1:
Firstly, veins on spinach leaves were removed until became 4 gram- pieces and then the pieces were placed into mortar. The 4 mg-leave tissue was chopped in ice cold mortar filled grinding solution by using pestle until as long as no solid parts remain. The solution was filtered by cheesecloth into a centrifuge tube to eliminate small soil that remained in it. In next stage, it was examined in centrifuge machine for 1 min at 200 xg. After centrifuge, supernatant was transmitted into a new centrifuge tube and the pellet was still unbroken cell and fragments. The remaining solution was centrifuged again at 1000 xg for 7 min. The supernatant was removed and the pellet was suspended by suspension solution after second centrifuge process because the pellet contains chloroplasts, heaver organelle than others in solution. For forming a solution, the mixture containing pellet and suspension solution were mixed by vortex machine. 8 ml acetone was added into a clear centrifuge tube with 1 ml chloroplast suspension to break structure of chloroplasts and achieve chloroplasts in chloroplasts. After adding 1 ml distilled water into centrifuge tube, the solution in tube was centrifuged for 5 min at 1000 xg. The sample was performed absorbance of supernatant at 658 nm. The measurement was applied at the formula mg of chlorophyll / ml of solution= absorbance / 34,5 to calculate mg of chlorophyll. Lastly, chlorophyll sample was diluted with suspension solution until the concentration of the sample was 0,01 mg/ml for next part of the experiment.
Part 2:
The diluted chloroplast suspension to 0,01 mg/ml was used for preparing different solutions according to table on the manual in 5 distinct tubes. Firstly, 3 ml K2HPO4/KH2PO4 buffer was filled into each 5 tubes. For first tube; 2 ml water was added into a tube. After that, chloroplast content was heated in water bath and was added into the same tube. For second tube; 1 ml water were added. 1 ml of heated chloroplast content was used for the second tube. At the third and fourth tubes, 3 ml active chloroplast and 1 ml water is filled. 1 ml water and 1 ml active were added into fourth tube. For last tube, Tube 5, was prepared by adding 1 ml active chloroplast and 1ml DCMU. These prepared solutions were performed at 600 nm by using spectrophotometer. Only Tube 1 was used as black in spectrophotometer. 1 ml DCPIP was dropped into Tube 2 and it was mixed gently, then it was measure the absorbance at 600 nm. Same process was realized for Tube 3, 4, 5. Only Tube 4 was covered with aluminium foil to not reach light for serving dark control. After measurements, Tube 2, 3, 5 was waited in ice under light for about 10 min. Then all tubes were placed into spectrophotometer to measure absorbance with 10 min-intervals; 10 min, 20 min and 30 min. At each step, results recorded.
Result:
Part 1: = * 10 dilution factor
Mg of chlorophyll= 0.2 mg
Note: The formula (absorbance = avg of absorbance- blank) wasn’t calculated because of spectrophotometer gave net solution.
For dilution : M1V1=M2V2 ; 0,2 mole/L*?ml=0,01 mole/L*10 ml ; ?ml=0,5 ml
Part 2:
0 min 10 min 20 min 30 min
Tube 1 0 0 0 0
Tube 2 0,419 0,408 0,393 0,403
Tube 3 0,393 0,390 0,386 0,385
Tube 4 0,387 0,391 0,380 0,380
Tube 5 0,397 0,409 0,386 0,388
Table 1-absorbance values
The reduce in the colour of dye was calculated from the formula Cr= C0(A0-AC)/A0 where ;
Cr : dye reduced in moles/liter
C0 : concentration of dye used (For DCPIP, it is 10-4 mole/liter)
A0 : Initial absorbance value of the mixture
Ac : Final absorbance value of the mixture
Interval/tubes Tube 2 Tube 3 Tube 4 Tube 5
0-10 0,00000262530 0,00000076336 -0,00000103359 -0,00000302267
0-20 0,00000620525 0,00000178117 0,00000180879 0,00000277078
0-30 0,00000381862 0,00000203562 0,00000180879 0,00000226700
Table 2- Moles/liter dye reduced of Tubes 2, 3, 4 and 5 at minutes 5, 10 and 20
Discussion:
Part 1:
At first stage of the experiment, we aimed isolation chloroplasts from leave tissue and calculating amount of chlorophyll by using absorbance with spectrophotometer. For achieving to get chlorophyll from chloroplasts, we used acetone to break structure of membrane of chloroplasts after cell fraction. We calculated mg of chlorophyll by using absorbance and formula. We multiplied with 10 for dilution factor because of added 8 ml acetone, 1 ml water and 1 ml chloroplast suspension. We got mg of chlorophyll as 0,2 mg. Lastly, we diluted to achieve 0,001 M for using next part of experiment. We used M1V1=M2V2 to calculate how we need distilled water for dilution. We used 0,5 ml water to dilute solution.
Part 2:
With change of content, we wanted to measure activity of chloroplasts. We used DCMU as photosynthetic inhibitor and DCPIP as a blue dye for measuring absorbance by using change colour. Reduced dye from blue to white forms by activity of photosynthesis. Spectrophotometer measures this colour change. Cr is reduction value of colour from blue to white. While turning into white, reduction value goes up and Cr value also increases too. According to this information, we don’t expect any change at Tube 2.Because chloroplasts in Tube 2 were denatured. When we investigate results, we can see a change in Tube 2. It means that chloroplasts are still active. We can think of heating wasn’t enough to denature chloroplasts. The results of Tube 3 and 4 are normal and in expected interval as values. Tube 4 had same values at second and third 10 min-calculations as difference than others. We have cover Tube 4 and it shouldn’t have been illuminated with light. The results show an error about Tube 4 and it was illuminated with light. This is an experimental error. Finally we investigated Tube 5. Photosynthesis has kept going for while then the value has decreased and this decreasing is expected. Because of without water, photosynthesis should have stopped slowly. Photosynthesis has processed for a while because at start tube has already 1 ml water. Negative values shouldn’t have existed. Likely the samples were deformed or there is a mistake at transferring into tubes. These values can’t be evaluated.
References:
Chloroplast, retrieved on 28.11.2016 (http://biology.tutorvista.com/animal-and-plant-cells/chloroplasts.html)
Photosynthesis, retrieved on 28.11.2016. Reece JB, et al. (2013). Campbell Biology. Benjamin Cummings. ISBN 978-0321775658.
Photosynthesis, retrieved on 28.11.2016. (http://www.rsc.org/Education/Teachers/Resources/cfb/Photosynthesis.htm)

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