Continue work from yesterday, but to observe the ADA kinetics with an inhibitor.
Creating the Adenosine Stock
- Add 0.1073 g of adenosine to a 10 ml volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a stock solution of 0.04 M.
- Add 10.00 μL of the 0.04 M adenosine stock to a 10 mL volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a final solution of 40 μM.
Enzyme Kinetics Measurement
- Add 3 mL of 40 μM adenosine solution to the cuvette and 30 μL of inosine.
- Start kinetics measurement:
- 1 ms integration
- 10 scan average
- Set "Save the first available scan every" to 15 seconds
- Set "Stop after this amount of time" to 10 minutes
- Set "File Type" to Tab Delimited
- Just before 1 minute, add 1.5 μL of 1 nM EHNA.
- 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.