BIOLOGY LABORATORY REPORT

Name: Nurseiit AlibekTutor: Dr. John CareyGroup: FLab partners name: Ismagulov GalymTitle: Investigating photosynthesis

IntroductionAlmost every plant takes water and CO2 (carbon dioxide) inside producing sugars or some complex substances. Waste product of the photosynthesis is oxygen. The total amount of energy of chemical bonds of the products is more than the total energy in the bonds of the raw materials, namely water and carbon dioxide. Consequently, this reaction appears to be an endergonic and it requires some external energy (energy can be taken from the sunlight). Chlorophyll (green substance) allow the plant to catch light energy that comes from the sunlight in order to produce sugars [1]. According to Kent (2000, 86), the term photosynthesis can be defined as the process of using sunlight to build up complex substances from simpler ones. Photosynthesis involves some complex processes and the simple equation for the photosynthesis can be written as follows: light and chlorophyll6CO2 + 12H2O C6H12O6 + 6O2 + 6H2O In addition, every chloroplast has two membranes, which are called the stroma. The stroma has enzymes, circular DNA and ribosomes, which can be used during the photosynthesis. Membrane sacs that are disk-like are the thylakoids. When several thylakoids are stacked together they form the granum. Also, these thylakoid membranes have chlorophyll (photosynthetic pigment). Moreover, photosynthesis is a very complex process and it has some complex reactions. Also, photosynthesis has two main stages. They are light-independent stage (dark stage) and light-dependent (light stage). Different books use different definitions to these stages. Obviously, light-dependent stage occurs only in the presence of light, whereas the light-independent stage does not require any light. The light-independent stage has the Calvin cycle, where the ATP and NADPH, which come from the light-dependent stage, are used in order to make some products, such as carbohydrates or lipids. This practical aims at demonstrating experimentally the link between the light-independent and light-dependent reactions. Early in 1937 year, the Robert Hill, who was a biochemist, found out the fact that isolated chloroplasts could produce oxygen when there is an illumination in the presence of an electron acceptor. When an electron acceptor (lets assume that electron acceptor is A) is reduced by protons and electrons, which come from water can be defined as Hill reaction and can be written as follows [2]: lightA + H2O AH2 + O2 chloroplasts

In this practical DCPIP (or the 2,6-dichlorophenol-indophenol), which is a blue dye, acts as an artificial electron acceptor. This artificial electron acceptor (DCPIP) has blue colour in its oxidized form, whereas in the reduced form it is colourless [2], [3]. Furthermore, centrifuge and spectrophotometer will be used during this practical. We have previously worked with the centrifuge, so we know how it works. However, the spectrophotometer is something new for us, so we should work with this apparatus very careful and also brief instructions that will be given by one of the lecturers or the TAs about the usage of this spectrophotometer should be taken into account.

Safety precautionsDuring this practical you should wear eye protective equipment (googles), gloves and lab.coat. Avoid skin contact with DCPIP, since it can stain. In addition, all procedures related with the centrifuge must be done in the presence of TAs or lecturers. In addition, we should keep the general safety precautions to avoid any emergency situation.

Materials and MethodsProcedureThis experiment was divided into two parts. Part 1 was isolating chloroplasts and part 2 was using the chloroplasts. In the first part (isolating the chloroplasts), three small green lettuce, cabbage or spinach were cut into small pieces using scissors. These small pieces were placed in prearranged cold mortar, which contained about 20 ml of cold isolation medium. Then, they were grinded rapidly and vigorously. After that, four layers of nylon were placed in the funnel and were wet with the cold isolation medium. This mixture was filtered through the funnel with the nylon into the beaker and then the filtrate was poured into pre-cooled centrifuge tubes, which were placed in the ice-water-salt bath before. After pouring the filtrate into pre-cooled centrifuge tubes, the edges of the nylon were gathered and were thoroughly wringed into the beaker and then transferred into the centrifuge tubes. The centrifuge tubes were checked whether they had the same volume of filtrate. Then, these tubes were centrifuged for some time in order to get a small pellet of chloroplasts. This procedure was performed in the presence of the teacher assistant. After the centrifuge, the liquid (supernatant) was poured off very carefully into the tube, because we must not lose any pellet during this experiment. This pellet was resuspended with 2 ml of the isolation medium using glass rod. After that, the procedure such as squirting in and out of a Pasteur pipette six or five times in order to get a uniform suspension. Finally, this leaf extract was stored in the ice-water-salt bath and was used as soon as possible in the second part of this practical. All of the solutions and apparatus, which were used in the first part, were kept cold in order to preserve the activity of enzymes. Also, the extraction was carried out as quickly as possible. In the second part (using the chloroplasts), the instructions were read before the second part starts. Five tubes were labelled and were set up according to the table, which is represented on the next page.

Table 1. The tubes were set up as follows:TubeLeaf extract(ml)Supernatant(ml)Isolation medium (ml)Distilled water (ml)DCPIP(ml)

10.55

20.55

30.55

40.55

50.55

* means that nothing has been added

After adding the DCPIP to the extract, the tube was shaken and the time was recorded. The tubes 1, 2 and 4 were placed on the place approximately 12-15 cm from a bright light (according to the manual 100 W). However, the tube 3 was placed in the dark place, namely under the table. All of the observations were clearly recorded into the logbook. At the same time, another procedure was performed. After a brief instruction about the use of the spectrophotometer, 5 ml of methanol was mixed with 200 l of the chloroplast suspension. Then, this solution was centrifuged at high speed (4000 rpm). Firstly, two small tubes (forgot the exact name of these tubes) were prepared. One of them contained the centrifuged mixture of the chloroplast suspension and the methanol, whereas another one contained only methanol. Then, the small tube contained the methanol was firstly placed into the spectrophotometer and only then the centrifuged mixture was placed into this spectrophotometer in order to measure the absorbance at 650 nm. In addition, the glass sides of these small tubes were checked because one side is muddy, so that this kind of thing might affect the result for the absorbance value. The same procedure was performed at 665 nm. Finally, the chlorophyll concentration was calculated.

Results:Table 2. Observation of the test tubes with different components.

TubeLeaf extract(ml)Supernatant(ml)Isolation medium (ml)Distilled water (ml)DCPIP(ml)

Observations

10.55After approximately 19 minutes there was a colour change from dark blue colour to the pale green.

20.55No change

30.55No change

40.55No change

50.55No change

* means nothing has been added

Measurement of the absorbance at different wavelengths:At 650 nm = 1.462At 665 nm = 1.000

DiscussionAccording to the obtained results, only first tube, which contained 0.5 ml of leaf extract and 5 ml of DCPIP, changed its colour from dark blue to the pale green after approximately 19 minutes. However, at the rest of the tubes there were no change, in other words colour of the solution did not change. The components in the first and third tubes were exactly the same. The main difference is that the first tube was placed under the light conditions, whereas the third tube was placed in the dark place. The change in colour in the first tube happens due to the production of the NADP2H, so after adding the DCPIP, which detects any production of reducing agents, the colour of the solution became pale green. Also, as was mentioned before there was no change in the third tube, since the production of the NADP2H depends on the light. So, this tube was placed in the dark place, consequently no light and no production of NADPH. As a result, there was no colour change as it was in the first tube. In addition, the fourth tube did not contain any DCPIP, consequently there was no change in colour and this was an expected result. The remaining tubes, namely second and fifth tubes also did not change their colour, since there was no leaf extract in these tubes.Moreover, the chlorophyll concentration can be calculated using this formula:Concentration (mg/ml) = 0.0255A650 + 0.004A665 The values for A650 and A665 are known. They are: A660 = 1.462; A665 = 1.000So, Concentration = (0.0255 x 1.462) + (0.004 x 1.000) = 0.04128 mg/ml.Moreover, let us compare again first and third tubes. First tube was under light conditions and it changed its colour from dark blue to the pale green, whereas third tube was under dark conditions and it did not change its colour. In both of the tubes there was a consumption of CO2. So, it can be concluded that CO2 has a little effect on the reducing capacity of the leaf extract. Also, CO2 was not involved in light-dependent reactions.There is a suggestion in order to investigate the effect of light intensity on the light-dependent reactions of photosynthesis. In order to do so, we should change the distance of the lamp or change the type of the light. Because if we place the lamp too close to the tubes, we might destroy some cells by overheating and of course it will affect the final results.

ConclusionTo sum up, during this practical the link between light-dependent and light-independent reactions were investigated. Also, only the first tube changed its colour, whereas the same tube, which was placed in the dark condition, did not change its colour. The fourt tube did not change its colour, since no DCPIP was added and obviously there was no artificial electron acceptor in this tube, so this result was an expected. Another thing is that second and fifth tubes also did not change their clour, since there was no leaf extract in these tubes. Moreover, the chlorophyll concentration was calculated using the data from the spectrophotometer. The concentration was 0.04128 mg/ml. In addition, in order to investigate the effect of the light intensity on the light-dependent reactions of the photosynthesis, I think we should change the distance of the lamp, measure this distance and see the results. Our results seem to be valid and reliable, since they correspond and converge with our general knowledge about the photosynthesis.

References1) Kent, Michael. 2000. Advanced Biology: Photosynthesis. UK: Oxford University Press.2) http://www.easternct.edu/~adams/Resources/Lab4%20Hill%20Rx.pdf3) http://www.marietta.edu/~spilatrs/biol309/labexercises/Photosynthesis.pdf

NAZARBAYEV UNIVERSITY