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Objective
To observe and measure ADA turnover kinetics in the presence of an inhibitor, EHNA.
adapted From Dr. Hartings.
Procedure
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.
Data
Notes
This area is for any observations or conclusions that you would like to note.
Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.
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