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Biology Coursework How Does Phosphate Concentration Inhibit

Biology Coursework How Does Phosphate Concentration Inhibit

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05/04/2014

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Biology Coursework
How does phosphate concentration inhibit the activity of bean sproutphosphatase?Phosphatase is an enzyme found in many organisms which hydrolyzesmolecules containing phosphate groups to release a phosphate ion. It is usefulin the human body to produce phosphate for many important biologicalmolecules such as ATP and phospholipids. Some phosphatase's work better atdifferent pH's. Alkaline phosphatase, found in places such as growing bones andteeth, is so named as the optimum pH is high. Acid phosphatase is has a lowoptimum pH and is found in the human kidney and the prostate gland.Phenolphthalein phosphate is a molecule containing phosphate thats reactioninto phenolphthalein and a phosphate ion is catalysed by phosphatase. In analkali solution the free phenolphthalein solution turns pink making a goodindicator of phosphatase activity.Here is an equation from the Science and Plants for Schools website to show thereaction:phenolphthalein ---------------------------------------> free phenolphthaleinphosphate (phosphatase enzyme) + phosphateMany enzymes are less active when the product they catalyze theformation of is present. This is called product inhibition. For example thepresence of galactose inhibits the activity of lactase. This is due to competitiveinhibition. Competitive inhibition occurs due to similar structure of the productand substrate. The substrate and the inhibitor therefore compete for binding tothe active site of the enzyme. As phenolphthalein phosphate is relatively similar in structure to phosphate it could be deduced that phosphate may act as acompetitive product inhibitor. Therefore my experiment will test this theory.I will carry out a trial experiment to find a suitable range of phosphateconcentration. The trial experiment will be almost identical to my experimenthowever I will use a large range of phosphate concentrations to give an idea of the scale appropriate for the main experiment. I will choose the concentrationsbased on the range that has effect on the end result. Because the trialexperiment will be very similar to my main experiment I will be able to identifyany procedural errors which could be eliminated. I will use phenolphthaleinphospate as the substrate for the phosphatase enzyme as the product,phenolphthalein, and causes alkaline solutions to turn pink therefore colorationof the solution will give an indication of enzyme activity. My independent variableis phosphate concentration. My dependent variable is coloration of my solutionwhich indicates phosphatase activity. Variables which may affect beansproutphosphatase activity need to be controlled, such as pH and tempurature. I willcontrol tempurature by placing my solution in a water bath whilst reaction is
 
taking place. pH may also be a variable that could affect enzyme activity and so Iwill control pH using a buffer solution. It is important to control these variables asthey can affect reaction rate so will affect my results. If these variables are notcontrolled it cannot identified clearly how phosphate concentration inhibits theactivity of beansprout phosphatase as other factors will influence the results.Alternative Hypothesis: The higher the concentration of phosphate present, theless the coloration of the end solution, indicating more enzyme inhibition. This isa one-tailed hypothesis.Null Hypothesis: Phosphate concentration will have no significant effect onenzyme activity so coloration of end solution will not vary significantly.
Equipment List
1 Measuring cylinder 5 Teat pipettes7 Test tubes1 250ml beaker Muslin Filter 1 Colorimeter 1 Cuvette30 C Water bath1 Mortar and PestleDistilled water 150 ml Phenolphthalein phosphateAbout 150 Beansprouts
Safety
Sodium carbonate is an alkali and therefore is an irritant to the skin and eyes.Contact should be avoided by wearing a lab coat and safety goggles. Freephenolphthalein is a suspected carcinogen and therefore contact should beavoided, especially ingested. However the small amounts present do notsurmount a large risk.
Pilot Procedure
For my test experiment I will use a large range (0-100%) of 0.3 phosphatesolution concentrations so as to get a sense of scale for the main experiment. Iwill prepare solutions of 0% 20% 33.3% 40% 66.6% 80% and 100% phosphatesolutions by using appropriate parts 0.3M phosphate solution and distilled water.I will extract phosphatase enzyme from the bean sprouts. Using a mortar andpestle and water and filter through muslin filter. I will then add phenolphthaleinphosphate to 7 test tubes and the phosphatase solution prepared. The test tubesmust then be placed in a 30 C water bath controlled and left to allow the reactionto take place. After 20 minutes I will take the test tubes out of the water bath,add 5cm of sodium carbonate solution to each test tube and mix. This solution
 
will turn pink if free phenolpthalein is present as it is alkali. I will then takereadings with the colourimeter to ensure readings are valid.In my pilot experiment the range of concentrations gave a good range of intensity of colourations and so I will use the same concentrations in my mainexperiment. However one issue raised by the pilot experiment was inaccuratereadings on the colorimeter due to the solution being too cloudy. This meant thatthe results were not valid. To rectify this problem in the main experiment I willuse the centrifuge to get a clearer solution. This should allow the colorimeter totake more valid measurements. I will also repeat the experiment twice more toimprove the reliability of the results and therefore the validity of any conclusionsthat can be drawn from them.
Plan
1. Prepare solutions of 0% 20% 33.3% 40% 66.6% 80% and 100% phosphatesolutions by using appropriate parts 0.3M phosphate solution and distilled water.The solution with 0% phosphate present acts as a control for my experiment.This is necessary both to test the null hypothesis and to compare my resultsagainst to provide context.2. Extract the phosphatase enzyme from the bean sprouts. To do this mix about150 bean sprouts with distilled water in a blender to create about 150cm of paste.3.Filter this through a double layer of muslin filter into a clean beaker. This willleave phosphatase solution ready to be centrifuged4. Prepare the phosphatase solution to go into the centrifuge by putting it incentrifuge tubes. Centrifuge for about 5 minutes. Remove from centrifuge.5. Prepare 7 test tubes by adding exactly 5cm of buffer solution to each testtube.6. Write 0%, 20%, 33.3%, 40%, 66.6%, 80% and 100% on 6 stickers.7. Label each test tube with a sticker.8. Accurately add 1cm of the corresponding phosphate solution to each testtube.9. Add 1cm of phenolphthalein phosphate to each test tube.10. Next add exactly 1cm of the phosphatase solution prepared in steps 2 and 3.Mix thoroughly.

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