Objectives
- Monitor the turnover kinetics of adenosine to inosine catalyzed by adenosine deaminase (ADA)
Kinetics
General Protocol:
- Prepare a 40μM solution of adenosine in 50mM phosphate pH 7.4 buffer.
- Note: This solution was prepared via dilution of a 0.0123M ADA stock solution
- Add 3mL of 40μM adenosine to a cuvette with a stir bar
- Note: All analysis should be carrier out in a temperature controlled environment. The temperature was kept at 25°C
- Allow the solution to reach a constant 25°C
- Begin taking absorbance spectra (take a spectrum every 15seconds)
- After 1min, add 30μL 1.1units/mL ADA
- Allow the reaction to run for 10 minutes, taking a spectrum every 15seconds.
Sample Preparation:
Table 1. Preparation of 0.0123M stock solution
| Mass of adenosine (g)
|
0.0165
|
| Volume of buffer(mL) |
5
|
Table 2. Preparation of 40μM adenosine solution
| Volume of stock(mL)
|
0.0098
|
| Volume of buffer(mL) |
2.9902
|
|
Project name
