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
|
UV-vis:
Figure 1. Corrected Absorbance spectra of the conversion of adenosine to inosine by ADA
- Using the molar absorptivity of inosine and adenosine determined on 2013/09/03, the conversion of adenosine to inosine can be represented as concentrations.
Table 3. Molar Absorptivity of Adenosine and Inosine
Adenosine
|
Inosine
|
13051 |
10793
|
Table 4. Concentration of Adenosine during the conversion to inosine
Time (s)
|
Concentration(M)
|
15 |
0.00004
|
30 |
|
45 |
|
60 |
|
75 |
|
90 |
|
105 |
|
120 |
|
135 |
|
150 |
|
165 |
|
180 |
|
195 |
|
210 |
|
225 |
|
240 |
|
255 |
|
270 |
|
285 |
|
300 |
|
315 |
|
330 |
|
345 |
|
360 |
|
375 |
|
390 |
|
405 |
|
420 |
|
435 |
|
450 |
|
465 |
|
480 |
|
495 |
|
510 |
|
525 |
|
540 |
|
555 |
|
570 |
|
585 |
|
600 |
|
Table 5. Monitoring the concentration of inosine during the conversion of adenosine to inosine by ADA
Time (s)
|
Concentration(M)
|
15 |
0.0000
|
30 |
|
45 |
|
60 |
|
75 |
|
90 |
|
105 |
|
120 |
|
135 |
|
150 |
|
165 |
|
180 |
|
195 |
|
210 |
|
225 |
|
240 |
|
255 |
|
270 |
|
285 |
|
300 |
|
315 |
|
330 |
|
345 |
|
360 |
|
375 |
|
390 |
|
405 |
|
420 |
|
435 |
|
450 |
|
465 |
|
480 |
|
495 |
|
510 |
|
525 |
|
540 |
|
555 |
|
570 |
|
585 |
|
600 |
|
|