Plan: The effect of the end product, phosphate, on the enzyme phosphatase
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The effect of the end product, phosphate, on the enzyme phosphatase 1. Plan Phosphatase enzymes release phosphates from a variety of substrates for synthesis of nucleotides, phospholipids, etc. They are found in both plant and animal tissues and can be classified as acid or alkaline depending on their optimum pH. In this experiment an acid phosphatase from potato was used. Hypothesis Since phosphate is a product of phosphatase activity, it may act as an end product inhibitor of the phosphatase enzyme, therefore slowing down the enzyme-catalyzed reaction. Background knowledge Enzymes are biological catalysts which speed up the metabolic reaction inside the cells. Enzymes are proteins and therefore can function because of their specific 3D shape. The active site is part of the molecule which allows the substrate to enter and form enzyme-substrate complex. If the shape of the active site changed, the substrate will not fit in, therefore the enzyme cannot function properly. The activity of enzyme is not only affected by temperature, pH, but also by enzyme inhibitors. These are the substances which can reduce the activity of enzymes. There are two types of enzyme inhibitors which refer to competitive and non-competitive inhibitor. Competitive inhibitor is a molecule which has a similar shape to the substrate, competing with the substrate for the active site of the enzyme and the inhibition can be either reversible or irreversible. The reversible inhibition means the inhibitors form a loose association with the enzyme and may become detached to the enzyme later, thus enabling the enzymes to function again. Irreversible inhibition means that the inhibitors combine permanently with the enzymes, making it impossible for substrates to react. Non-competitive inhibitor is a molecule which does not enter the active site of the enzyme but changes its shape by entering the other part of the enzyme, leaving the enzyme unable to work. However, Cells always contain many natural inhibitors which control the rate of metabolism and known as the reversible non-competitive inhibitor.
Then place the two test tubes opposite to each other in the centrifuge, put the lid back and centrifuge for bout 5 minutes. After that, pour the supernatant into a clean test tube. This is used as the enzyme suspension. Label 15 test tubes A1-A5, B1-B5, C1-C5 and add 5 cm3 buffer from beaker 1 to tube A1, B1, C1 by using the same syringe; then using the same syringe add 5cm3 buffer from beaker 2 to tubeA2, B2, C2 and continue this same procedure step wise to beaker5. Add 1cm3 of the 1% phenolphthalein phosphate to each tube by a 1 cm3 syringe. Then use a kettle to make some hot water and then mix with cold water in the 1000ml beaker. Use a thermometer to stir and add any either cold or hot water until the temperature keeps at steady 30?. Then add 1 cm3 of enzyme suspension to each tube and mix them well with 5 different stirring rods, e.g. one rod stir A1,B1,C1and the other one stir A2,B2,C2 and so on. Place them together in the 30? water bath and at the same time start the stop clock. During the incubation, add any hot water if temperature drops. After being incubated for 20 minutes, put all the test tubes out of the water bath and place them back on the rack. Add 5cm3 of 10% sodium carbonate solution to each tube and mix as before. The tubes can now be stored in a fridge until the next day if required. Then record the color of each test tube and measure the intensity of the pink colour using a colorimeter with a 550nm filter. Record the absorbance. Present them in a table with headings. Then calculate the average absorbance for each concentration of sodium phosphate by using the value of A1, B1 and C1 for 0M, A2,B2 and C2 for )0.05M and continue this procedure to A5, B5 and C5.
First I will make 7 buffer 5 solution each contains sodium carbonate with concentration 0.0, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45 mol/cm respectively. Label them from A to D. Second I will choose a big potato to extract the enzyme phosphatase in the same way of last experiment. Then I will prepare 4 sets of test tubes each contains 7 test tubes. Label them from A1 to D7. Then add buffer A to test tube sets A, buffer B to test tube sets B and so on. Add 1cm of the 1% phenolphthalein phosphate to each tube by a 1 cm syringe. Stir the enzyme suspension well and add 1cm of it into each test tube and mix them well. Then put all the test tubes in the same water bath of 30?. After 20 min incubation, and assistant and I will take them out of the water bath and add sodium carbonate to each test tube at the same time to stop the reaction. Then use a colorimeter with precise division to quantify the color change in each test tube(from colorless to pink). Calculate the average of A1 B1 C1 and A2 B2 C2 and so on. Draw these 7 values on a graph paper and also the error bar. Deducing from the results of last experiment, I predict that the graph pattern would be a straight line with negative gradient, and between the sodium phosphate concentration 0.35 and 0.45 mol/cm, the rate of reaction will be 0. Investigation 2 To investigate whether the result of the investigation, the end product effect is only specific to phosphatase in the potato, or has a commonness to all the enzymes, I will carry out the following two experiments. One will be the same process as the investigation of end product effect but use the beansprouts to extract the enzyme phosphatase. I predict that the rate of reaction decreases as the concentration of sodium phosphate increases and the pattern of the graph will be the same as the potatoes'.
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Here's what a teacher thought of this essay
This is a very well written report that demonstrates a good understanding of scientific processes and methods.
1. The background knowledge section is well written and concise but should include references.
2. The variables section is well thought through.
3. The method section should be simplified and restructured.
4. The data is presented well.
5. The conclusion is detailed but again needs references.
6. The evaluation is the strongest section in the report and shows good statistical knowledge.
***** (5 stars)
Marked by teacher Luke Smithen 01/05/2013
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