To observe and measure ADA turnover kinetics in the absence of an inhibitor. This work will be the basis of our comparison to ADA-AuNP turnover studies.
From Dr. Hartings.
Creating the Adenosine Stock
- Add 0.1071 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 9.98 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.
- 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 30 μL of 0.01 units/mL ADA.
Adenosine deaminase (ADA) stock
- 1.1mg (24.0 units in 1 mg) in 25mL of buffer --> 1.1 units/mL ADA
ADA for experiments
- 100uL of stock ADA and 900uL buffer --> 0.11 units/mL ADA