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An Investigation on the Effect of Inhibitor Concentration on Reactions involving Acid Phosphatase.

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Introduction

An Investigation on the Effect of Inhibitor Concentration on Reactions involving Acid Phosphatase. Katherine Pears CONTENTS Introduction and Hypotheses: Introduction...................................................................Page 3 Pilot Study Summary.......................................................Page 3 Hypotheses...................................................................Page 3 Theory..........................................................................Page 4 Methods: Apparatus List................................................................Page 9 Pilot Study.....................................................................Page 9 Method.......................................................................Page 12 Choice of Techniques....................................................Page 13 Controls......................................................................Page 15 Risk Analysis...............................................................Page 16 Analysis: Data Tables...............................................................Page 17 Graphs........................................................................Page 19 Discussion and Evaluation: Explanation..................................................................Page 22 Error analysis...............................................................Page 22 Limitations...................................................................Page 24 Suggestions for Further Study.......................................Page 24 Appendices Bibliography.................................................................Page 26 An Investigation of Inhibitor Concentration on Reactions involving Acid Phosphatase. INTRODUCTION AND HYPOTHESES Introduction I have decided to investigate the effect of altering the concentration of an inhibitor, on an enzyme catalysed reaction. I will be investigating this with the reaction aided by the enzyme acid phosphatase, in which phenolphthalein and phosphates are released. The inhibitor being used will be sodium dihydrogen phosphate. Pilot studies lead to this idea as they were successful and the most interesting theory was provided using this variable. Pilot Study Summary I carried out a pilot study which involved demonstrating the effect of an inhibitor on the reaction involving the breakdown of phenolphthalein. This developed into an investigation on the effect of inhibitor concentration on an enzyme catalysed reaction. Null Hypothesis There will no effect of changing the amount of inhibitor used. The % transmission will remain constant for all solutions despite varying concentration of inhibitor. Alternative Hypothesis As the concentration of sodium dihydrogen phosphate is increased the rate of reaction will decrease. This will be indicated by a decrease in % transmission as concentration of inhibitor decreases. THEORY The reaction that I am investigating is: In this reaction the enzyme is acid phosphatase. The definition of an enzyme is a globular protein which acts as a biological catalyst, increasing rate of chemical reactions, without itself being permanently changed [7]. The enzyme acid phosphatase assists reactions in which phosphate compounds are broken down releasing phosphates. In this case the compound phenolphthalein diphosphate is used to act as an indicator of the rate of reaction. ...read more.

Middle

I obtained the following results: Results for Pilot 3 Concentration (M) % transmission (blue filter) 0.10 90.00 0.09 88.00 0.08 88.00 0.07 88.00 0.06 88.00 0.05 86.00 0.04 86.00 0.03 86.00 0.02 84.00 0.01 79.00 0.00 39.00 As expected there was not enough variation in my results to carry out a worth while investigation. I then decided to vary the concentration between 0.00M and 0.01M rather than 0.1M. From this pilot study I decided on the range of inhibitor concentration I would be using. Method 1. Place a 250mL glass beaker on a balance and tare, weigh 30g of mung beans into the beaker. 2. Place mung beans and 30mL of water in a pestle and mortar. Add a small amount of sand and grind into a pulp. 3. Place muslin cloth over mouth of 50mL beaker. Use an elastic band to hold cloth in place if necessary. 4. Squeeze pulp through a muslin cloth into a 50mL beaker. Divide the liquid evenly into a 4 centrifuge tubes use a 25mL measuring cylinder to achieve equal volumes. Centrifuge at 60 r.p.m for 5 minutes. 5. Pour supernatant into a clean 50mL beaker and dispose of pellet. 6. Place 11 boiling tubes in a boiling tube rack. 7. Use a 1 cm3 pipette to measure 1 cm3 of 1% phenolphthalein diphosphate solution, add 1 cm3 to each boiling tube. When measuring volumes of liquids measure from bottom of meniscus and check this at eye level. 8. Use two 10 cm3 pipettes to make up different concentrations of buffer pH5 and 0.01M sodium dihydrogen phosphate. Make following concentrations: Concentrations for Experiment Concentration (M) Volume of 0.01M inhibitor (cm3) Volume of buffer 5 (cm3) 0.010 10.00 0.00 0.009 9.00 1.00 0.008 8.00 2.00 0.007 7.00 3.00 0.006 6.00 4.00 0.005 5.00 5.00 0.004 4.00 6.00 0.003 3.00 7.00 0.002 2.00 8.00 0.001 1.00 9.00 0.000 0.00 10.00 9. ...read more.

Conclusion

I had enough space to carry out the experiment well. As I was only slightly limited by time all that may have been sacrificed is attention to detail. If i had had access to professional apparatus such as a spectrophotometer, then the accuracy in my experiment may have improved. Suggestions for Further Study Obviously to gain a further understanding for this topic I would carry out tests at more concentrations of inhibitor. I would especially aim to gain more results for the concentrations of inhibitor between 0.01M and 0.02M. I would do this as this is where the graph levelled off. By obtaining these values I would find where the maximum inhibition. To obtain these concentrations I would make up the following: Inhibitor Concentration (M) Volume of buffer 5 (cm3) Volume of 0.02M Inhibitor (cm3) 0.010 5.0 5.0 0.011 4.5 5.5 0.012 4.0 6.0 0.013 3.5 6.5 0.014 3.0 7.0 0.015 2.5 7.5 0.016 2.0 8.0 0.017 1.5 8.5 0.018 1.0 9.0 0.019 0.5 9.5 0.020 0.0 10.0 To these concentrations I would add the constants to each boiling tube as in the experiment I carried out in this investigation. These were 1cm3 of substrate, 1cm3 of enzyme and 5cm3 of alkali. I would then obtain these results in the same was as in this investigation. Also to further my knowledge on this topic I would carry out the experiment in the same way but change the variable of pH. This would show me what the optimum pH for the enzyme acid phosphatase is. I have researched this but to be sure which is the best I would carry out an experiment to show this. To do this I would not use any inhibitor, as this slows down the reaction and in this case this is unnecessary as inhibitor concentration would not be investigated. I would add the same volume of buffer, enzyme and substrate to each boiling tube. The variable would be the pH of the buffer. To vary this I would add the same volume of different pH buffers to each boiling tube. ...read more.

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