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Need help on 13-14
13) Read through the procedures. Notice that youre asked not to add the enzyme immediately when preparing the reaction mixtures. What do you think would happen if you did add the enzyme immediately? How would this affect your readings/results? 14) Look at the table on p. 8, then answer the questions below. a) In which tube would you expect to observe a greater reaction rate: Tube 1 or Tube 22 Explain your answer. In which tube would you expect to observe a greater reaction rate: Tube 1 or Tube 3? Explain your answer. b) In which tube would you expect to observe a greater reaction rate: Tube 2 or Tube 4? Explain your answer. e) d) To determine whether Inhibitor X is a competitive- or non-competitive inhibitor, you would have to compare the results from Tube to the results from Tube e) If Inhibitor X is a non-competitive inhibitor, explain what you would expect to see in the tubes you selected in part d.
ACTIVITY 5: Ihe Effect of Inhibitors on Enzyme Activity MATERIALS NEEDED (per group) . 6 spectrophotometer tubes(cuvettes), At least 5.0 ml of 4.0 mM ONPG . At least 1.5 ml of 3.3 FCC units/ml lactase enzyme solution . At least 1.5 ml of Inhibitor X .At least 1.5 ml of Inhibitor Y e 10 mM Sodium Phosphate Buffer, pH 7.0 .Squirt bottle containing dH O P1000 pipette and tips .P200 pipette and tips . Container for disposing used pipette tips . Kimwipes PROCEDURE 1. Using the table shown below, prepare 6 different reaction mixtures. everything except the enzyme solution; you will add the enzyme at a later step. Prepare your reaction mixtures in the smaller test tubes that fit into the spectrophotometer. Change tips after transferring each reagent so that your stock For now, add solutions do not become contaminated. Use a grease pencil to label the top of each tube; writing towards the middle of the tube will disrupt the spectrophotometer reading. Tube Vol. of 4mM Vol. of Vol. of Vol. of Vol. of 3.3 Tot. Rxn ONPGInhibitor X Inhibitor Y 10 mM FCC Vol. Phosphate units/ml Buffer solution2000 enzyme 500 μ1 1000 ul 500 1000 μι 1300 μι l 200 ul 2000 μι ! 80011 200 μι 2000 11 60011 200 μΙ 2000 μι | 100 1 700 μι 5 500 l 700 μι 600 μι 1 200 μι 2000 μι 200 11 2. If you havent already, prepare the spectrophotometer. Do this by setting it to measure absorbance (not transmittance) at 450 nm. Mix the tube containing your enzyme solution. Then, place the appropriate volume of enzyme solution into Tube #1. Mix the tube by flicking it with the fleshy part of your forefinger. Next, place the tube into the spectrophotometer and press the zero button so it gives a reading of zero absorbance at the start of the run. Record the absorbance on the data table provided on p. 14 of this lab exercise every 30 seconds for 2 minutes. 3. 4, 5. 6. Remove the tube from the spectrophotometer. Repeat Step #3 with Tubes #2-6. When you finish, empty and thoroughly rinse all the spectrophotometer tubes. Plot the data, and determine the reaction rate (change in OD units per minute) for each
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