The objective of today's lab is to observe and measure ADA turnover kinetics in the presence of, EHNA, an inhibitor.
The following protocol was taken from Matt Harting's notebook
- Make a new 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
- Go to Dr. Hartings lab for enzyme kinetics measurements.
- Add 2μL of EHNA to the 40μM solution of adenosine buffer
- Add 3mL of adenosine solution to the cuvette
- Start your kinetics measurement
- 1ms integration (on front panel)
- 10 scan average (on front panel)
- Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
- Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
- Set "File Type" to Tab Delimited
- Give the files a directory and a name
- Click accept
- Just before 1 minute add 30ul of 1.1u/mL ADA
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
- Graph of the changes in concentration from adenosine into inosine over time.
- Adding EHNA appears to slow down the conversion of adenosine into inosine