Enzyme Pre-Lab Writeup

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Bzne96

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Hi, I need a 2 paragraph Pre-Lab writeup for my enzyme lab in my Biology class. You will need to READ the 4 pages of the lab that I will attach to this and do the pre lab assignment which I will attach as well on the last attachment which has to be only 2 paragraphs. Please answer all the questions on the pre lab assignment (Last Attachment I will post) and do not plagiarize from anywhere or do not use any outside sources for any of the questions besides the lab pages I post. If you have any questions please let me know! Thank you!

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STATISTICAL HYPOTHESES Statistical analysis involves a comparison of the expected results derived from a predictione tot the observed results represented by the experimental data. For the sample experiment above, we predicted that pH would affect enzyme activity. From this prediction, we can formulate the following hypotheses: Biological hypothesis: pH has an effect on the rate of starch digestion by fungal amylase. Null hypothesis (Ho): There is no significant change in amylase activity as the buffer pH changes. Any deviation observed is due to chance. Alternative hypothesis (Ha): There is a significant change in amylase activity as the buffer pH changes. pH has a significant effect on amylase activity. We will use a formal statistical test to determine whether to reject or fail to reject the null hypothesis. In this case, since the biological hypothesis is consistent with the alternative hypothesis, if we reject the null hypothesis, the biological hypothesis is supported. EXPERIMENTAL DESIGN The Effect of Temperature on Enzyme Activity Today, we will NOT investigate the effects of pH. Instead, we will explore the effects of temperature on enzyme activity. Our question of interest is whether a 20°C decrease in temperature leads to a significant change in amylase activity. The class will develop an experiment to answer this question. Materials Available fungal amylase starch solution buffer, pH 7.5 ice bath (4°C) and room temperature (24°C) environments After we run the experiment, we will use a new statistical test called a t test to assess the significance of the results. To employ this test, we must first formulate both a biological hypothesis and a related pair of statistical hypotheses for our experiment exploring the effect of temperature on enzyme activity. 46 Constructing a standard curve and measuring an unknown sample We will prepare a standard curve by measuring the absorbance of starch solutions of known concentration treated with IKI. We will measure a range of samples, then plot starch concentration against absorbance on a graph. We can then determine the amount of starch present in an unknown sample by finding its absorbance value on the standard curve. 0.4 Absorbance (580 nm) 0.2 0 0.2 0.4 Starch concentration (mg/ml) Figure 2. Standard curve There are three reaction tubes in your rack. The blue tubes contain starch solutions treated with IKI. The yellow tube is the blank, and contains everything in those tubes except starch. This tube is used to zero the spectrophotometer, so that any absorbance observed depends only on the amount of starch present, not on any other reagents (e.g. buffer, IKI). A B SPECTRONIC 200E Home Menu SPEC 2009 Modern Interface SPEC 20 D+ Emulation OD600 Admnistration A 10.00 On ). Figure 3. Spectronic 200 (A) controls and (B) sample chamber 1. Set the wavelength: Use the I knob to select 580 nm. Turning the knob will change the wavelength by increments of 10. For precise adjustment, hold it down while turning. 2. Zero the spectrophotometer: Wipe the blank tube and place it in the slot at the left side of the chamber as shown. Press the 0.00 button. The purpose of the blank is to provide a reference for the lightest condition possible. 3. Measure absorbance: Remove the blank and insert the known sample. Record the absorbance shown and concentration values for each group in the table below. 4. Use this data to construct your own standard curve on the graph below. 43 Sample Experiment: Effect of pH on Enzyme Activity The following sample experiment explores the effect of pH on amylase activity. This is NOT the question you will ask in lab, but it will be a useful guide for your own experimental design. Consider the hypothesis: Amylase has an optimal pH where it exhibits greatest activity. This leads to a prediction: The further pH is from this optimum, the lower its activity. The hypothesis reduces the experiment to two variables. The independent variable, pH, will be modified to observe its effect on the dependent variable, starch digested. Table 2. Sample Experimental Design tube (-) control 2 3 4 5 (+) control starch 0 ml 1 ml 1 ml 1 ml 1 ml 1 ml amylase 1 ml 1 ml 1 ml 1 ml 1 ml 0 ml buffer 2 ml pH 7 1ml pH 5.5 1ml pH 6.5 1ml pH 7.5 1ml pH 8.5 2ml pH 7 total (ml) | 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml Table 3. Sample Data Set A B A - B tube independent variable (pH) initial starch (mg/ml) absorbance (580 nm) 1 OU AWN 7.5 5.5 6.5 7.5 8.5 7.5 0.0 0.5 0.5 0.5 0.5 0.5 0.05 1.40 0.24 0.5 1.5 1.5 starch remaining based on curve (mg/ml) 0.0 0.5 0.185 0.28 0.5 0.5 starch digested from equation (mg/ml) 0.0 0.0 0.315 0.22 0.0 0.0 A plot of the results would look like this: 0.4 0.3 Starch digested (mg/ ml) 0.2 0.1 0.0 5.5 6.5 pH 7.5 8.5 Figure 5. Relationship Between pH and Amount of Starch Digested What conclusion(s) can you draw from these results? 45 Enzyme Activity Note: There is a pre-lab writeup for this lab due at the start of your lab session (see p.47). OBJECTIVES OS 1. Describe what an enzyme is and how factors such as temperature affect enzyme activity 2. Describe the reaction that takes place when the enzyme amylase encounters starch 3. Explain what a standard curve is and why one is necessary in this experiment 4. Use a spectrophotometer and understand how it works 5. Use statistical methods to evaluate a hypothesis at a given confidence level INTRODUCTION Organisms cannot rely entirely on spontaneous reactions to produce all of the materials necessary for life. Such reactions occur too slowly, and are not responsive to an organism's needs. As a solution to this problem, cells employ biological catalysts known as enzymes to mediate certain reactions. Like all catalysts, enzymes speed up reactions without being consumed in the process. Each enzyme acts on a specific molecule or set of molecules, called its substrate(s). The molecules that result from the reaction are called products. An enzyme facilitates a reaction so that substrates are converted into products more quickly. Enzyme Substrate(s) Product(s) The enzyme amylase helps disassemble the polysaccharide starch, the primary energy storage molecule in plants. Human salivary glands make amylase to begin digestion of dietary polysaccharides in the mouth. Amylase catalyzes the breakdown of starch into the disaccharide maltose, a simple sugar that can be used to generate the energy needed to power cellular work. As this reaction proceeds, the amount of starch decreases and the amount of maltose increases, while the amount of amylase remains constant. Amylase Starch Maltose Amylase, like most enzymes, is a protein whose structure is determined by the sequence of amino acids from which it is built. Each enzyme is built to function optimally at physiological conditions of pH and temperature. Since enzyme function is highly dependent on structure, any change in these variables that alters the conformation of the enzyme can either enhance or destroy its activity. This will greatly influence the rate of the reaction. For most enzymatic reactions, a temperature decrease of 10°C leads to an approximate 2-fold decrease in activity. This change reflects a 2-fold reduction in the number of molecules with thermal energy equivalent to the activation energy required for the reaction to occur. We will explore the rate of reaction of amylase over a temperature range of 20°C to determine whether its activity shows the expected decrease. 41 MEASURING ENZYME ACTIVITY A Amylase activity can be quantified by measuring a change in the amount of either substrate (starch) or product (maltose) as a function of time. We will measure its activity by following the disappearance of starch using the iodine test. With this test, a deep blue color indicates starch is present, and the amber color of iodine itself indicates starch is absent. The intensity of the blue color is related to the amount of starch present, and can be determined quantitatively using a spectrophotometer. What is a spectrophotometer and what does it do? A material appears colored because some wavelengths of light are absorbed by it, some pass through it, and others are reflected. For example, the sky appears blue because the atmosphere reflects light of the wavelengths we perceive as blue, while it allows most others to pass through undisturbed. A spectrophotometer measures the amount of light of a specific wavelength absorbed by a sample. The darker a solution appears, the greater its absorbance. A spectrophotometer works by passing light through a filter that can be adjusted to control the wavelength emitted to the test sample. This filtered light passes through the sample and hits a phototube on the other side. The less light that is absorbed by the sample, the more hits the phototube, and the lower the absorbance reading. Since your eyes are nearly as sensitive as a spectrophotometer, observe your solutions as you run the experiment to confirm that the absorbance readings are reasonable. What your eye cannot do, and a spectrophotometer can, is quantify how much color is present. Be aware that anything foreign in the sample or on the tube (e.g. bubble, fingerprint) will block the passage of light through the sample and give a false absorbance reading. Experimental procedure inclo 100 We will use a spectrophotometer to quantify the results of an iodine test to measure the amount of starch remaining after reaction with amylase. We will employ a standard curve to convert the absorbance level into actual units of starch, in this case mg/ml. amylase starch maltose 1 1 20 +IKI w V blue solution V quantify intensity of color with spectrophotometer V use a standard curve to convert to mg/ml starch V +IKI V yellow solution Figure 1. Flowchart of the experiment Enzyme Activity Note: There is a pre-lab writeup for this lab due at the start of your lab session (see p.47). OBJECTIVES OS 1. Describe what an enzyme is and how factors such as temperature affect enzyme activity 2. Describe the reaction that takes place when the enzyme amylase encounters starch 3. Explain what a standard curve is and why one is necessary in this experiment 4. Use a spectrophotometer and understand how it works 5. Use statistical methods to evaluate a hypothesis at a given confidence level INTRODUCTION Organisms cannot rely entirely on spontaneous reactions to produce all of the materials necessary for life. Such reactions occur too slowly, and are not responsive to an organism's needs. As a solution to this problem, cells employ biological catalysts known as enzymes to mediate certain reactions. Like all catalysts, enzymes speed up reactions without being consumed in the process. Each enzyme acts on a specific molecule or set of molecules, called its substrate(s). The molecules that result from the reaction are called products. An enzyme facilitates a reaction so that substrates are converted into products more quickly. Enzyme Substrate(s) Product(s) The enzyme amylase helps disassemble the polysaccharide starch, the primary energy storage molecule in plants. Human salivary glands make amylase to begin digestion of dietary polysaccharides in the mouth. Amylase catalyzes the breakdown of starch into the disaccharide maltose, a simple sugar that can be used to generate the energy needed to power cellular work. As this reaction proceeds, the amount of starch decreases and the amount of maltose increases, while the amount of amylase remains constant. Amylase Starch Maltose Amylase, like most enzymes, is a protein whose structure is determined by the sequence of amino acids from which it is built. Each enzyme is built to function optimally at physiological conditions of pH and temperature. Since enzyme function is highly dependent on structure, any change in these variables that alters the conformation of the enzyme can either enhance or destroy its activity. This will greatly influence the rate of the reaction. For most enzymatic reactions, a temperature decrease of 10°C leads to an approximate 2-fold decrease in activity. This change reflects a 2-fold reduction in the number of molecules with thermal energy equivalent to the activation energy required for the reaction to occur. We will explore the rate of reaction of amylase over a temperature range of 20°C to determine whether its activity shows the expected decrease. 41 MEASURING ENZYME ACTIVITY A Amylase activity can be quantified by measuring a change in the amount of either substrate (starch) or product (maltose) as a function of time. We will measure its activity by following the disappearance of starch using the iodine test. With this test, a deep blue color indicates starch is present, and the amber color of iodine itself indicates starch is absent. The intensity of the blue color is related to the amount of starch present, and can be determined quantitatively using a spectrophotometer. What is a spectrophotometer and what does it do? A material appears colored because some wavelengths of light are absorbed by it, some pass through it, and others are reflected. For example, the sky appears blue because the atmosphere reflects light of the wavelengths we perceive as blue, while it allows most others to pass through undisturbed. A spectrophotometer measures the amount of light of a specific wavelength absorbed by a sample. The darker a solution appears, the greater its absorbance. A spectrophotometer works by passing light through a filter that can be adjusted to control the wavelength emitted to the test sample. This filtered light passes through the sample and hits a phototube on the other side. The less light that is absorbed by the sample, the more hits the phototube, and the lower the absorbance reading. Since your eyes are nearly as sensitive as a spectrophotometer, observe your solutions as you run the experiment to confirm that the absorbance readings are reasonable. What your eye cannot do, and a spectrophotometer can, is quantify how much color is present. Be aware that anything foreign in the sample or on the tube (e.g. bubble, fingerprint) will block the passage of light through the sample and give a false absorbance reading. Experimental procedure inclo 100 We will use a spectrophotometer to quantify the results of an iodine test to measure the amount of starch remaining after reaction with amylase. We will employ a standard curve to convert the absorbance level into actual units of starch, in this case mg/ml. amylase starch maltose 1 1 20 +IKI w V blue solution V quantify intensity of color with spectrophotometer V use a standard curve to convert to mg/ml starch V +IKI V yellow solution Figure 1. Flowchart of the experiment Constructing a standard curve and measuring an unknown sample We will prepare a standard curve by measuring the absorbance of starch solutions of known concentration treated with IKI. We will measure a range of samples, then plot starch concentration against absorbance on a graph. We can then determine the amount of starch present in an unknown sample by finding its absorbance value on the standard curve. 0.4 Absorbance (580 nm) 0.2 0 0.2 0.4 Starch concentration (mg/ml) Figure 2. Standard curve There are three reaction tubes in your rack. The blue tubes contain starch solutions treated with IKI. The yellow tube is the blank, and contains everything in those tubes except starch. This tube is used to zero the spectrophotometer, so that any absorbance observed depends only on the amount of starch present, not on any other reagents (e.g. buffer, IKI). A B SPECTRONIC 200E Home Menu SPEC 2009 Modern Interface SPEC 20 D+ Emulation OD600 Admnistration A 10.00 On ). Figure 3. Spectronic 200 (A) controls and (B) sample chamber 1. Set the wavelength: Use the I knob to select 580 nm. Turning the knob will change the wavelength by increments of 10. For precise adjustment, hold it down while turning. 2. Zero the spectrophotometer: Wipe the blank tube and place it in the slot at the left side of the chamber as shown. Press the 0.00 button. The purpose of the blank is to provide a reference for the lightest condition possible. 3. Measure absorbance: Remove the blank and insert the known sample. Record the absorbance shown and concentration values for each group in the table below. 4. Use this data to construct your own standard curve on the graph below. 43 Sample Experiment: Effect of pH on Enzyme Activity The following sample experiment explores the effect of pH on amylase activity. This is NOT the question you will ask in lab, but it will be a useful guide for your own experimental design. Consider the hypothesis: Amylase has an optimal pH where it exhibits greatest activity. This leads to a prediction: The further pH is from this optimum, the lower its activity. The hypothesis reduces the experiment to two variables. The independent variable, pH, will be modified to observe its effect on the dependent variable, starch digested. Table 2. Sample Experimental Design tube (-) control 2 3 4 5 (+) control starch 0 ml 1 ml 1 ml 1 ml 1 ml 1 ml amylase 1 ml 1 ml 1 ml 1 ml 1 ml 0 ml buffer 2 ml pH 7 1ml pH 5.5 1ml pH 6.5 1ml pH 7.5 1ml pH 8.5 2ml pH 7 total (ml) | 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml Table 3. Sample Data Set A B A - B tube independent variable (pH) initial starch (mg/ml) absorbance (580 nm) 1 OU AWN 7.5 5.5 6.5 7.5 8.5 7.5 0.0 0.5 0.5 0.5 0.5 0.5 0.05 1.40 0.24 0.5 1.5 1.5 starch remaining based on curve (mg/ml) 0.0 0.5 0.185 0.28 0.5 0.5 starch digested from equation (mg/ml) 0.0 0.0 0.315 0.22 0.0 0.0 A plot of the results would look like this: 0.4 0.3 Starch digested (mg/ ml) 0.2 0.1 0.0 5.5 6.5 pH 7.5 8.5 Figure 5. Relationship Between pH and Amount of Starch Digested What conclusion(s) can you draw from these results? 45 STATISTICAL HYPOTHESES Statistical analysis involves a comparison of the expected results derived from a predictione tot the observed results represented by the experimental data. For the sample experiment above, we predicted that pH would affect enzyme activity. From this prediction, we can formulate the following hypotheses: Biological hypothesis: pH has an effect on the rate of starch digestion by fungal amylase. Null hypothesis (Ho): There is no significant change in amylase activity as the buffer pH changes. Any deviation observed is due to chance. Alternative hypothesis (Ha): There is a significant change in amylase activity as the buffer pH changes. pH has a significant effect on amylase activity. We will use a formal statistical test to determine whether to reject or fail to reject the null hypothesis. In this case, since the biological hypothesis is consistent with the alternative hypothesis, if we reject the null hypothesis, the biological hypothesis is supported. EXPERIMENTAL DESIGN The Effect of Temperature on Enzyme Activity Today, we will NOT investigate the effects of pH. Instead, we will explore the effects of temperature on enzyme activity. Our question of interest is whether a 20°C decrease in temperature leads to a significant change in amylase activity. The class will develop an experiment to answer this question. Materials Available fungal amylase starch solution buffer, pH 7.5 ice bath (4°C) and room temperature (24°C) environments After we run the experiment, we will use a new statistical test called a t test to assess the significance of the results. To employ this test, we must first formulate both a biological hypothesis and a related pair of statistical hypotheses for our experiment exploring the effect of temperature on enzyme activity. 46 PRE-LAB WRITEUP Read through the section “The Effect of Temperature on Enzyme Activity" on the previous page. Provide the following information in 1-2 typed paragraphs due at the start of lab (10 pts). Form a biological hypothesis to test in lab that relates temperature to enzyme activity. State the null hypothesis and alternative hypothesis that you will statistically evaluate. Identify the single variable you will change between experimental tubes 2 and 3. Make a prediction about which tube (2 or 3) will exhibit a higher level of enzyme activity. Identify the component you will omit from the positive control tube, which is expected to have the maximum possible absorbance. Identify what you will omit from the negative control, expected to have zero absorbance. Explain why you will need to add additional buffer to control tubes 1 and 4. Identify which tube (1-4) you will place in the spectrophotometer to zero the absorbance. Include a table that identifies the volume of each component you will add to tubes 1-4. Use Table 2 from the Sample Experiment as a model. Table 4. Your Experimental Design tube # 1: negative control 2 3 4: positive control temperature room temperature room temperature starch (ml) amylase (ml) buffer (ml) total (ml)
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