User:Eleni N. Kalivas/Notebook/CHEM-571/2013/10/09

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Objective

The objective of today's lab is to observe and measure ADA turnover kinetics in the presence of, EHNA, an inhibitor.

Protocol

The following protocol was taken from Matt Harting's notebook

  1. Make a new 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
  2. Go to Dr. Hartings lab for enzyme kinetics measurements.
    1. Add 2μL of EHNA to the 40μM solution of adenosine buffer
    2. Add 3mL of adenosine solution to the cuvette
    3. Start your kinetics measurement
      1. 1ms integration (on front panel)
      2. 10 scan average (on front panel)
      3. Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
      4. Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
      5. Set "File Type" to Tab Delimited
      6. Give the files a directory and a name
      7. Click accept
      8. Just before 1 minute add 30ul of 1.1u/mL ADA

EHNA stock 5mg EHNA in 1mL DMSO ---> 15.9mM EHNA (1.9μL)(15.9mM EHNA)=(10mL)(C2). C2 ---> 3μM EHNA The reaction samples will contain roughly 1nM EHNA

Data

  • Graph of the changes in concentration from adenosine into inosine over time.

Adenosine Concentrations vs. Time.png

  • Adding EHNA appears to slow down the conversion of adenosine into inosine