Chapter 2. Analyzing Enzymes and Enzyme Kinetics II

Learning Objectives

Lab 3—Specific Enzymatic Analysis
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General Purpose

Conceptual

  • Be able to relate changes in different factors to changes in the rate of enzyme-catalyzed reactions.
  • Be able to formulate testable hypotheses, including H0 and Ha.
  • Be able to design and conduct experiments to test your hypotheses.
  • Be able to clearly present the results and conclusions of an experiment to others.

Procedure

Have your lab instructor review the specific methods and techniques of your written procedure before you begin your experiment. You will use the spectrophotometer to measure absorbance at 500 nm every 30 seconds over a three-minute period as you did in the previous laboratory.

  1. If the spectrophotometer is not already on then turn the spectrophotometer on and allow it to warm up for 15 minutes.
  2. Prepare the appropriate blank solution or blank solutions.
  3. Adjust the wavelength of the spectrophotometer to 500 nm. Set the mode to transmittance. Properly zero and blank the spectrophotometer using the blank solution prepared in step 2 (refer to Appendix A if needed). Don’t forget to clean the surface of the tube with a Kimwipe before placing it in the spectrophotometer.
  4. Prepare your reaction tubes as you have outlined in your lab notebook. Remember that once the substrate and enzyme are together in the same tube the reaction will proceed very quickly. Do not combine these ingredients until you are ready to measure the absorbance.
  5. Set the mode of the spectrophotometer to absorbance. Place the “reaction tube” in the spectrophotometer sample holder and record the absorbance in the table you prepared in your laboratory notebook. This initial reading will be the absorbance reading for time “0.”
  6. Measure the absorbance every 30 seconds for 180 seconds and record these data in a table in your lab notebook.
  7. Remove the reaction tube from the spectrophotometer and repeat this process (steps 3–6) for all of your reaction tubes. For some experiments you will need to re-blank the spectrophotometer with a different blank solution between treatments.

Clean-Up Procedure

  1. Contents of BLANKS AND REACTION TUBES can be poured down the sink drain.
  2. Markings on ALL tubes should be removed before the tubes are placed in the plastic box on the laboratory instructor’s cart.
  3. Dispose of gloves in the biohazard waste container in the classroom.
  4. Return safety glasses to their designated place.