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I wanted to find out whether the addition of zinc sulphate, and gradual increase in concentration had a detrimental effect on the ability of chloroplasts to reduce DCPIP (therefore meaning that Zinc had inhibited photosynthesis as DCPIP will only be reduced when photosynthesis is uninhibited and occurring as normal). I made up 9 solutions in order of increasing concentration of Zinc Sulphate and timed how long it took for DCPIP to be fully reduced. I concluded that the addition of Zinc does have an effect on the reduction of DCPIP, but as the Zinc concentration rises past around 3 dm3 it has little to no effect. The inhibitor made it take longer for DCPIP to be fully reduced.
Research and Rationale
DCPIP (2,6-dichlorophenolindophenol) is used to measure the rate of photosynthesis in green plants. One of the two stages of photosynthesis is the light dependant reaction. This ends with the electron transport chain and the reduction of NADP+. However, when DCPIP is involved it also gets reduced at this stage; therefore, we can measure the progress of photosynthesis by using DCPIP, a colorimeter and the knowledge that DCPIP is blue when oxidised and colourless when reduced.
http://en.wikipedia.org/wiki/File:DCPIP-reduction-2D-skeletal.pngIn the following diagram the reduction of DCPIP can be seen:
The process of photosynthesis is made up of two separate processes known as the light dependant and the light independent reaction. In the first step - the light dependent reaction - a pair of electrons from chlorophyll is raised to a higher energy level by the light energy absorbed. They're then accepted by an electron acceptor and passed along a chain of carriers situated in the thylakoid membrane. Photophosphorylation then occurs (when energy that has been released is used to convert ADP and inorganic phosphate into ATP.) The electrons then pass into another chlorophyll molecule and eventually pass to NADP with the hydrogen from H2O to form reduced NADP. When DCPIP is present, it takes the place of NADP as an electron acceptor. The ATP and reduced NADP are now ready to be used in the light independent reaction to make the final carbohydrate from CO2.
The light dependant reaction is as follows: CO2 combines with a five carbon compounds called ribulose biphosphate, catalysed by the enzyme ribulose biphosphate carboxylase. The six carbon compound formed is highly unstable and breaks down instantaneously into two 3 carbon molecules of glycerate 3-phosphate. This is then reduced to form a 3 carbon sugar phosphate called glyceraldehyde 3-phosphate. The hydrogen for this reduction comes for the reduced NADP from the light dependent reactions. Two out of every 12 GALPs formed are involved in the creation of a hexose sugar while ten out of every 12 GALPs are involved in the recreation of ribulose biphosphate to allow the cycle (called the Calvin Cycle) to continue.
The Hill Reaction was discovered by Robert Hill in 1937 when he found that isolated chloroplasts can generate oxygen when a suitable electron acceptor is present and light is available. This was important to the study of photosynthesis because it allowed us to work out that the source of electrons was in fact, water and that the oxygen that was formed came from the water and not from carbon dioxide. The formal definition of the Hill Reaction is the reduction of an electron acceptor by electrons and protons from water, with the formation of oxygen when chloroplasts are exposed to a light source  .
Ribulose biphosphate carboxylase-oxygenase (Rubisco) is the enzyme that catalyses the start of the photosynthesis reaction, it is one such enzyme that may be affected by the addition of metal ions. Rubisco catalyses the initial reaction in the Calvin cycle of photosynthesis.
There are many enzymes involved in the complex process of photosynthesis. Enzymes make reactions go thousands to millions of time faster, every reaction that occurs within cells has its own enzyme; they require very specific conditions and the presence of heavy metals such as zinc can cause the enzyme to function less effectively. An enzyme has an active site, where its substrate will fit and also an allosteric site. The allosteric site is a place on the enzyme where a non-substrate molecule can bind, and alter the shape of the active site and so mean the substrate can no longer fit. Thus certain reactions cannot be catalysed, inhibiting photosynthesis in chloroplasts.
"In isolated barley chloroplasts the presence of two millimolar ZnSO4 inhibits the electron transport chain activity of photosystem II as measured by the photo reduction of dichlorophenolindophenol O2 evolution and chlorophyll Î± fluorescence."
"These observations suggest that Zn inhibits electron flow at the oxidising side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide"
"No inhibition of photosystem I dependent electron transport by three millimolar ZnSO4 is observed however with concentrations of ZnSO4 above 5 millimolar photosystem I activity is partially inactivated"
Heavy metals are found in many different aspects of human activity. Heavy metals are used in the production of paint, electronic materials, batteries and most importantly pesticides. The metal ion particles in pesticides could potentially be absorbed into the plants they are used on and affect the allosteric sites on the enzymes inhibiting their respiration.
An increased concentration of zinc will have a significant effect on the ability of DCPIP to be reduced in the electron transport chain of photosynthesis.
An increased concentration of zinc will have no significant effect on ability of DCPIP to be reduced in the electron transport chain of photosynthesis.
Initial trial and study
In my initial trial I decided on a series of volumes of Zinc Sulphate that came to 2ml including the addition of distilled water. I deemed this a suitable amount based on the fact that the chloroplast/buffer solution mixture equalled 5ml. My first trial was more difficult than anticipated because the starting colour of my solution would be different each time affecting the absorption of light affecting the end outcome. I managed to improve this situation by adding more buffer solution as I thought the colour change may be down to pH differences between each solution. Following that, I made my chloroplast/buffer solution mixture up to 10ml with more buffer solution.
Weigh out 4g of Spinach leaves and cut them up into small pieces, leaving out the stalks, since the leaves contain more chloroplast than the stalks do.
Place the cut up Spinach leaves into a cold mortar and pestle with 15ml of cold buffer solution and some grinding sand.
Grind the Spinach until the mixture becomes a mesh.
Filter the mesh of Spinach leaves through a cheese cloth, funnelling the obtained solution into 2 centrifuge tubes.
Make sure there are equal amounts in each tube and place them into a centrifuge for 10 minutes.
Decant the supernatant from the obtained pellet at the bottom of the tubes, add 10 ml of cold buffer solution to the pellet and mix together.
Transfer this new solution into an ice bath at 0-5 Degrees Celsius, this is the chloroplast solution.
Prepare a cuvette for the colorimeter; add 0.5ml of the chloroplast solution and 3ml of distilled water. Using the wavelength of light 680nm measure the filled cuvette, this will be the reference light absorption.
Prepare cuvettes with different concentrations of DCPIP, Distilled H20, Zinc Sulphate and chloroplast solution, as soon as they are added take absorbance readings and record the value, continue to take readings every 30 seconds for 4 minutes for each different cuvette.
Take repeats of this experiment and plot a graph of the results, the % of DCPIP reduction over 4 minutes on the y axis and the zinc concentration on the x axis.
30 Spinach leaves
The Spinach leaves contain the chloroplast needed for the DCPIP to be reduced.
500cm3 of Buffer solution
I needed a lot of Buffer since it was needed in every reaction I did, the Buffer solution guaranteed that the colour of the measured solution did not change, as Buffer solutions maintain the pH, so it doesn't change and affect the experiment.
500cm3 of Distilled water
Distilled water was used to create the different levels of stock of Zinc Sulphate, changing the concentrations, meaning I wouldn't have to order in every different concentration of Zinc Sulphate required for my experiment.
500cm3 of DCPIP
DCPIP is the indicator in this experiment, it is used in every measured solution so a lot is needed so I didn't run out, and I had more than enough for 1 trial and 3 repeats.
500cm3 of Zinc Sulphate
The Zinc Sulphate is the inhibitor in this experiment so I needed a lot to cover the entire experiment. The amount I chose was more than enough,
The colorimeter measures the percentage absorption of light at a wavelength of 680nm.
Holds the solution to be measured in the colorimeter.
The Centrifuge pushes all of the solid in the solution to the bottom, creating a pellet at the bottom, this is the chloroplast.
These are used to hold the solution to be centrifuged.
The cheese cloth is used to filter out any solid out of the mesh of spinach leaves.
The spatula simply helps to grind up the pellet, helping it to mix with the buffer solution.
The ice bath is used to freeze the chloroplast and buffer mixture, to stop any reaction taking place (possible colour change) so when you want to start the reaction it will be completely fresh and unchanged.
10cm3 pipette and a 1cm3 pipette
The pipettes are used to accurately measure the amount of each reactant in the experiment, to reduce percentage error i.e. more accurate than a measuring cylinder.
The test tubes were used to hold the different concentrations of chloroplast to distilled water.
These are used to keep the chloroplast solution active while out of the colorimeter to keep the reaction going.
The chemicals that I used in my experiment were Zinc Sulphate, DCPIP and Buffer solution.
Zinc Sulphate is harmful if ingested or if it makes contact with the eyes, causing irritation and possible permanent damaging effects to the eyes. Chronic exposure problems include: Prolonged and regular contact with Zinc Sulphate on the skin can cause dermatitis and eye conjunctivitis. Breathing in Zinc Sulphate can cause bronchoconstriction, which in turn can lead to chronic asthma. Acute exposure problems include: Gastrointestinal disturbance may occurring if ingested and the inhalation of Zinc Sulphate can cause sinus problems and oesophageal irritation.
Although Zinc Sulphate is not flammable it can however, melt and produce toxic zinc and sulphur oxide toxic fumes. However, I only used small quantities of it and was careful to wash my hands between experiments and repeats so as to ensure I didn't get it in my eyes. My exposure to zinc sulphate was limited and not prolonged and so I thought it safe to use.
DCPIP had very low risk, minor possibilities of problems that could occur. There was the possibility of mild irritation in contact with the skin or via inhalation in the throat. These risks I judged very small and deemed DCPIP safe to use. I made sure to always wash my hands and avoid touching my face and eyes during the course of the experiment
Independent Variable: Concentration of Zinc Sulphate
Dependent Variable: Effect on light absorbance by colorimeter
Photosynthesis is primarily affected by light intensity, temperature and pH. To maintain a constant light intensity, I used a desk lamp placed a set distance away from my apparatus. This ensured that lack of light or too weak a light intensity could be ruled out as a variable. To ensure a constant pH, I added a buffer solution which meant that the pH never deviated from neutral by a significant degree.
The anomalies I had in my raw data are highlighted in blue; I omitted the anomalies when calculating the average % change over 4 minutes. The anomalies may have been caused by overexposure to UV light accidentally by passing the chloroplast solution under a lamp. I may also have not adjusted the colorimeter correctly between readings and that caused the reading to be different. Human error is also a possibility; I may have simply read the colorimeter incorrectly or not pushed the button and recorded a previous result instead.
I used Spearman's rank because it allows you to test the relationship of one variable against the other, the correlation between the independent and dependant variable can be seen clearly so you can accurately and confidently reject or accept your null hypothesis.
Data Set 1 (Concentration of Zinc Sulphate)
Data Set 2 (Average % Change over 4 minutes)
Total = 312
The results for Spearman's Rank can vary between -1 and 1 with -1 equalling negative correlation, 0 equalling no correlation and +1 equalling positive correlation (between variables.)
My critical value for a significance level of 5% was 0.648 therefore I can reject my null hypothesis (Which was: An increased concentration of zinc will have no significant effect on ability of DCPIP to be reduced in the electron transport chain of photosynthesis) at the 95% confidence limit which means that I can be 95% confident that my obtained results are not down to chance. I can accept my experimental hypothesis of: An increased concentration of zinc will have a significant effect on the ability of DCPIP to be reduced in the electron transport chain of photosynthesis. My correlation is -0.891 which shows me that as the Zinc concentration increases, the Hill Reaction in photosynthesis is inhibited (which we can see because DCPIP is blue when oxidised and colourless when reduced and so as photosynthesis becomes inhibited, less DCPIP is reduced and less lights is absorbed through the still blue solution.)
Trends and Patterns
There is a downward/negative trend of light absorption as time increases. There appears to be no correlation between increased Zinc concentration and light absorption past 3cm3 of Zinc Sulphate in the solution.
Even before I manipulated the data to show mean percentage decrease in light absorption over 4 minutes, I could see that there was an obvious and significant downward trend in the light intensity readings as zinc concentration increased and time went on. Following data manipulation it was obvious that zinc was a significant negative correlation between increasing zinc concentration and light absorbed.
Explanation of Results
When photosynthesis is occurring uninhibited, the DCPIP will go colourless as it is reduced through accepting a pair of electrons from the oxygen atom off water. However when you add zinc, it binds to the allosteric site on the enzymes involved in photosynthesis, such as ribulose biphosphate and changes the shape of the active site, meaning the enzyme can no longer accept its substrate and so photosynthesis is inhibited and the DCPIP will remain blue, meaning less light gets through. This explains the decreasing trend of my results. Less light is being absorbed at 240 seconds than at the beginning of the experiment. So photosynthesis starts off well (i.e. more light gets through the solution because it's lighter blue/colourless) but soon gets inhibited and so less light gets through (i.e. it is blue now).
I definitely would make modifications to the method and overall procedure in which I carried out my experiment. I would control the amount of light the chloroplasts received throughout the experiment because it greatly affected the rate of photosynthesis during the 4 minutes and thus the % light absorbed by the colorimeter.
In conclusion, there is significant statistical data to say that, as the concentration of zinc rises, the average percentage change decreases up till a concentration of 4x10-4mol dm3 of Zinc Sulphate where it levels off. I can reject my null hypothesis at the 95% confidence limit.