Objective
- Complete Activity Assay for ADA
Procedure
- UVProbe was opened.
- Window > 1. Kinetics
- Methods Icon
- Wavelength: 265nm
- Duration: 300 seconds (5minutes)
- OK
- Shimadzu CPS-Controller was set to 25°C.
- wait for the temperature to raise to 25°C
- place the sample in the cell and click start.
- Phosphate buffer was used as a blank and was used to create the baseline.
- Kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table below).
- The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).
- 5mM adenosine stock solution was too concentrated and exceeded the capacity of the UV-Vis therefore it was diluted:
- 150 uM adenosine stock solution was used; (stock) 150uL 5mM stock + 850 uL buffer =750 uM ->use .6mL new stock for ADA assay
- 200 uM adenosine stock solution was used: (stock) 200 uL 5mM stock+800 uL buffer = 1000uM ->use 0.6mL new stock for ADA assay
- This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
- This table is the updated table of the volumes and concentrations of solutions that will be added in the cuvette.
Data
Concentration of Adenosine (uM)
|
Average Velocity over 30s (nm/sec)
|
200 |
0.00021
|
100 |
0.000183333
|
40 |
0.00012
|
16 |
0.0001
|
6.4 |
0.000047
|
2.56 |
0.000033
|
1/[adenosine] (1/uM)
|
1/[velocity] (sec/nm)
|
0.005 |
4761.904762
|
0.01 |
5454.545455
|
0.025 |
8333.333333
|
0.0625 |
10000
|
0.15625 |
21276.59574
|
0.390625 |
30303.0303
|
-1/Vmax |
6163.1
|
Vmax |
0.000162256 nm/sec
|
-1/Km |
-0.093
|
Km |
10.75 uM
|
|