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