Enzyme Acid Phosphatase Activity

Enzyme Acid Phosphatase Activity - Acid Phosphatase...

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Acid Phosphatase Activity: The Effects of Temperature, pH, and Substrate, Enzyme, and Inhibitor Concentration Anna Krajec October 29, 2011 Lab Section 01
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Tables and Graphs Part A – Standard Curve for p-Nitrophenolate Part B – Effect of Temperature Part C – Effect of Enzyme Concentration Part D – Effect of pH Part E – Effect of Substrate Concentration Part F –Effect of Phosphate Inhibitor Part G – Effect of Fluoride Inhibitor
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Part H – Combined Plot
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Calculations Sample Calculation for μ M p-nitrophenolate (part A) Sample Calculation for moles p-nitrophenolate produced per hour (parts B, C, and D) Sample Calculation for moles p-nitrophenolate produced per hour (parts E, F, and G) Determining Types of Inhibition Because the Phosphate inhibitor line intersects with the non-inhibited enzyme line near the y- axis, it is a competitive inhibitor. The two reactions have equal V MAX values and different K M values. Since the Fluoride inhibitor line intersects with the Substrate Concentration line near the x-axis, it is a noncompetitive inhibitor. Its reaction’s K M value is the same as that of the reaction without the inhibitor, but the V MAX values are different.
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Pre-Lab Questions 1. How will increasing enzyme concentration affect the rate of an enzyme-catalyzed reaction? Increasing enzyme concentration will increase the rate of an enzyme-catalyzed reaction because there is more enzyme present to act upon the substrate, thus moving the reaction along quicker. 2. What effect will increasing the temperature have on the rate of an enzyme-catalyzed reaction? Increasing the temperature will initially increase the rate of an enzyme-catalyzed reaction, because the molecules move faster and therefore occupy the active sites more quickly. However, after a certain temperature (optimum temperature), the rate will begin to decrease, because high temperatures denature enzymes. 3. How will the rate of the enzyme-catalyzed reaction in this experiment be followed? Describe what will be measured to determine the rate. The rate of the enzyme-catalyzed reaction will be followed using a fixed-time assay. After 15 minutes, the solution will be poured into NaOH in order to kill the enzyme and react with the product. In basic solution, p-nitrophenol (the product of hydrolysis of the substrate) absorbs light very strongly at 405 nm and turns the solution a yellow color. The more yellow the solution, the further along the reaction. The rate of the reaction will be determined by the amount of p-nitrophenolate generated, measured by the absorbance at 405 nm of the solution after 15 minutes. 4. What is the name of the synthetic substrate for acid phosphatase? What are the names and structures of the products of the reaction? The artificial substrate that will be used is p-nitrophenyl phosphate (NPP), which hydrolyzes to p-
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This note was uploaded on 12/10/2011 for the course BCH 5045 taught by Professor Guy during the Fall '08 term at University of Florida.

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Enzyme Acid Phosphatase Activity - Acid Phosphatase...

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