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, pH, and temperature on the activity of the enzyme amylase. Amylase is found in the saliva of many animals, including humans, that utilize starch as a source of food. Starch, the principal reserve carbohydrate stores of plants, is a polysaccharide composed of a large number of glucose monomers joined together. Amylase is responsible for the preliminary digestion of starch. In short, amylase breaks up the chains of glucose molecules in starch into maltose, a two-glucose-unit compound. Further digestion of this disaccharide requires other enzymes present in pancreatic and intestinal secretions. To help us follow the digestion of starch into maltose by salivary amylase, we will take advantage of the fact that starch, but not maltose, turns a dark purple color when treated with a solution of I2KI (this solution is normally yellow-amber in color). In the following experiments, the rate of disappearance of starch in different amylase concentrations allows a quantitative measurement of reaction rate. Recall that the rate of appearance of the product (in this case, maltose) would give the same information, but the starch test is simpler. EXERCISE 1. The Influence of Enzyme Concentration on the Rate of Starch Digestion Materials test-tube rack 8 standard test tubes sharpie several test plates flask of distilled or DI water I2KI solution 1% starch solution 2 1-mL calibrated pipettes calibrated 5-mL pipette disposable pasteur pipettes pipette pump pH 6.8 buffer solution 5-mL graduated cylinder 1% amylase solution
Introduction In this experiment you will vary the concentration of the enzyme amylase to determine what effect the variation will have on the rate of the reaction. You will make serial dilutions of the amylase resulting in a range of enzyme concentrations. For serial dilutions, you will take an aliquot (sample) of the original enzyme and dilute it with an equal amount of water for a 1:1 dilution (50 % of the original concentration). You will then take an aliquot of the resulting 1:1 solution and add an equal amount of water for a 1:3 dilution of the original concentration. You will continue this series of dilutions until you have four different amylase concentrations. Question Pose a question about enzyme concentration and reaction rate.
(2) Quickly remove 1 drop of the mixture with a disposable pasteur pipette.063% amylase) 2. Add 5 mL amylase solution from tube D2 to tube D3 and mix. (1)Using a clean 1-mL pipette. Add 5 mL amylase solution from tube D3 to tube D4 and mix. Procedure 1. a. b. Transfer 1 mL of the dilution from tube D1 into tube E1. the reaction has begun! 2 . One team member should immediately record the time. c. Mix by rolling the tubes between your hands.5 mL of the 1% starch solution to tube E4 and mix by rolling the tube between your hands.5% amylase) d. (This is a 1:15 dilution which results in 0. Set these tubes aside.Hypothesis Hypothesize about the effect of changing enzyme concentration on the rate of reaction. Add 5 mL amylase to tube D1 and mix by rolling the tube between your hands. f. (This is a 1:7 dilution which results in 0. Number a second set of four standard test tubes E1 through E4. (This is a 1:1dilution which will result in 0. and add it to a drop of I2KI in the first compartment on the test plate (time). Prediction Predict the results of the experiment based on your hypothesis (if/then). Beginning with tube E4. g. Add 5 mL amylase solution from tube D1 to tube D2 and mix. Prepare the amylase dilutions as follows: a. Add 20 drops of pH 6. c. add 0. e. Add 1 or 2 drops I2KI to each compartment of a test plate. Transfer 1 mL of the dilution from tube D2 into tube E2.125% amylase) f. h. Using the 5 m-L graduated pipette. b. Transfer 1 mL of the dilution from tube D3 into tube E3. One the dilutions have been prepared you will prepare the experimental test tubes. d. Transfer 1 mL of the dilution from tube D4 into tube E4. This is time 0.25% amylase) e. *Remember. when the enzyme and substrate are together. You will use a separate row for each concentration of amylase.8 buffer solution to each of the tubes (E1-E4). add 5 mL distilled water to each test tube. (This a 1:3 dilution which results in 0. Number four standard test tubes D1 through D4.
5. 5. Table 1. Time of Starch Disappearance in Different Concentrations of Amylase. 7.063 EXERCISE 2. (4) Repeat steps 1 through 4 for the other three concentrations (tubes E3. 6. Hypothesis Hypothesize about the rate of activity of amylase at various pHs. E2 and E1). The Effect of pH on Amylase Activity Materials test-tube rack 1% amylase solution 7 standard test tubes I2KI solution several test plates 1% starch solution sharpie pipette bulb 7 buffer solutions (pH = 5.25 0. 8) 3 5 mL calibrated pipettes disposable pasteur pipettes Introduction The environmental factor pH can influence the three-dimensional shape of an enzyme.5. Tube % Amylase Time of Starch Disappearance (sec) E1 E2 E3 E4 0. 6.(3) Sample the reaction mixture at 10-second intervals. Every enzyme has an optimum pH at which it is most active. In this experiment you will determine the optimum pH for the activity of amylase. Record the time required for the digestion of the starch. continue until a blue color is no longer produced and the I2KI solution remains yellow-amber (indicating the digestion of all the starch). 3 .5.5 0. 7. each time using a new compartment of the test plate. Question Pose a question about pH and reaction rate.125 0.
5 7 7. This is time 0. 4. Procedure 1. Using a clean 1-mL pipette. Table 2. 3. Sample the reaction mixture at 10-second intervals. add 2. 5.5 6 6. add 1. and add it to a drop of I2KI in the first compartment on the test plate (time). Repeat steps a through b for the other six pH values. *Remember. when the enzyme and substrate are together. pH 6 through pH 8. b. continue until a blue color is no longer produced and the I2KI solution remains yellow-amber (indicating the digestion of all the starch). Label seven standard test tubes as follows: pH 5. Using a clean 5-mL graduated pipette. Starting with the pH 5 tube: a.Prediction Predict the results of the experiment based on your hypothesis (if/then).5 mL of the 1% starch solution to tube pH 5 and mix by rolling the tube between your hands.5 8 4 . each time using a new compartment of the test plate. 2. Time of Starch Disappearance in Different pH Environments for the Enzyme Amylase. Quickly remove 1 drop of the mixture with a disposable pasteur pipette.5 mL amylase solution to each tube and mix by rolling the tubes in your hands. Record the time required for the digestion of the starch. pH 5.5. One team member should immediately record the time. d. Add 1 or 2 drops of I2KI into the compartments of several rows of the test plate. pH Time of Starch Disappearance (sec) 5 5. use a 5-mL graduated pipette to add 5 mL of the appropriate buffer to each test tube. Once the test tubes have been labeled. the reaction has begun! c.
Using the 5-mL calibrated pipette. a 10° rise in temperature results in a two-to threefold increase in the rate of a particular reaction. the integrity of proteins can be irreversibly denatured. This means that substrates collide more frequently with enzyme active sites. The Effect of Temperature on Amylase Activity Materials 8 standard test tubes test-tube rack 2 5 mL calibrated pipettes 2 1 mL calibrated pipettes disposable pasteur pipettes pipette pump sharpie 1% starch solution buffer solution (pH = 6. add 4 mL DI water to each tube. Prediction Predict the results of the experiment based on your hypothesis (if/then). 5. add 2 mL of the 1% starch solution to each tube. at high temperatures. depending on the structure of the enzyme. Procedure 1. Hypothesis Hypothesize about the rate of activity of amylase at various temperatures. However. The activity of enzymes is dependent on the proper tertiary and quaternary structures. Generally. Place the test tubes as follows: Tube 1: 80°C water bath Tube 2: 37°C water bath Tube 3: test-tube rack at room temp Tube 4: beaker of crushed ice (4°C) 5 . partly because increased temperatures speed up the motion of molecules. 4. 3. Using a clean 5-mL pipette. Question Pose a question about temperature and reaction rate.EXERCISE 3.8) 1% amylase solution flask of DI water water bath at 37° C water bath at 80° C beaker of crushed ice for ice bath I2KI solution Introduction Chemical reactions accelerate as temperature rises. the optimum temperature for activity. may vary. Number four standard test tubes 1 through 4. Add 1 mL of pH 6.8 buffer to each tube. 2. therefore.
Continue adding mixture drops to new wells of I2KI at 30-second intervals until a blue color is no longer produced and the I2KI solution remains yellow-amber (indicating that all the starch is digested). when the enzyme and substrate are together. 10.6. Repeat steps 9 and 10 for the other reaction mixtures (mix tubes 2 and 2A and test. *Remember. and place as follows (do not mix together the solutions in the two sets of tubes until instructed to do so): Tube 1A: 80°C water bath Tube 2A: 37°C water bath Tube 3A: test-tube rack at room temp Tube 4A: beaker of crushed ice (4°C) 7. record the time (this is time 0). etc. Let all eight tubes sit in the above environments for 10 minutes. If within 10 minutes there is no color change. 8. mix 3 and 3A and test. Use the 1-mL calibrated pipette to add 1 mL amylase to each tube. the reaction has begun! 9. (oC) Time of Starch Disappearance (sec) 1 2 3 4 80o 37o 22o 4o 6 .). Table 3. mix tubes 1 and 1A. terminate the experiment with that particular reaction mixture. Leaving the tubes in the above environments as they are being tested. Time of Starch Disappearance in Different Temperatures for the Enzyme Amylase Tube Temp. Number and mark a second set of standard test tubes 1A through 4A. You should have one tube of amylase and one of starch at each temperature. and use a disposable pipette to immediately add 1 or 2 drops of the mixture to a drop of I2KI on the test plate. Fill several rows of the test plate with 1 or 2 drops of I2KI per compartment. 11.
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