“An Enzyme Kinetics Experiment for the Undergraduate Organic Chemistry Laboratory” R.J. Olsen, J.A. Olsen and G.A. Giles Instructor’s Notes: •
We do this experiment late in the Organic II course, after students have been trained in the routine use of the NMR spectrometer, and approximately when amino acids, peptides and proteins are being discussed in class. Sign-up sheets are posted and students sign up in partnerships for approximately two hours of instrument time outside of their regular lab period. They are strongly encouraged to prepare the substrate solution in advance so that the instrument time can be spent on the instrument.
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The experiment could also be done as a “dry lab” in which NMR data is provided to the students.
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There have been two primary sources of failure observed in our experience with this experiment: Failure to properly prepare the substrate solution, especially when adjusting the pH. To avoid this problem, the substrate solution could be prepared in advance by the instructor. Failure to mix the enzyme suspension with the substrate solution in the NMR tube. Simply inverting the tube may not be sufficient since D2O doesn’t flow in a 5 mm NMR tube the way organic solvents do, and the enzyme suspension may form a “plug” at the top of the tube. It is necessary to agitate the tube fairly vigorously.
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The derivation of the substrate concentration formula is an interesting exercise for the algebraically inclined.
Safety: •
The usual caution should be exercised when working with the sodium hydroxide solutions. Rubber gloves are recommended.
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The usual precautions should be exercised when working in the vicinity of a strong magnetic field.
