Continue ADA kinetics assay
Goals
- We picked up from yesterday and continued with the Kinetic Assay protocol
- We made solutions of gold in our sample of adenosine deaminase. This procedure and concentrations can be found in Michael's notebook
- We also began the process of conducting dialysis on our samples of ADA in order to remove the salts from the samples
Procedure
- For all information regarding the making of the gold and ADA solutions and the dialysis of the ADA, please see Michael's notebook.
- From the spectras collected yesterday, we looked at the absorbances of inosine and adenosine at two specific wavelengths, 235nm and 265 nm. These absorbances were used to calculate the molar absorptivity using Beers Law A=εbc
- For inosine, at λ235, ε= 1400; λ265 ε= 6000
- and for Adenosine at λ235, ε= 18566; λ265 ε= 13029
(For full calculations, please see Mary's notebook)
- based on these calculations, which were checked multiple times, the greatest differential was seen to be at λ235, where adenosine has an extremely high molar absorptivity and comparatively, the absorptivity for inosine was relatively low. Based on this fact, in the following samples, we monitored the kinetics of the reaction at a wavelength of 235. in these samples, there was adenosine, ADA and phosphate buffer present. in the catalysis of this reaction, ADA being an enzyme would eat up the adenosine and convert it into inosine. as a result, on our spectra, we expected to see the absorbance of the sample to decrease as time went by.
- The following is what each of our assays was supposed to be based on the protocol:
| Assay
|
Phosphate buffer [mL]
|
Adenosine [mL]
|
ADA solution [µL]
|
| 1 |
2.7 |
0.3 |
15
|
| 2 |
2.7 |
0.3 |
25
|
| 3 |
2.7 |
0.3 |
40
|
| 4 |
2.7 |
0.3 |
50
|
- However, all of our trials continue to not only have an absorbance of over 1 but at the specific wavelength we were monitoring, the absorbances were increasing as time went by, and not decreasing and we had no indication of what was causing this fluctuation. We continued to try various different samples by varying the parameters such as decreasing the amount of adenosine in the cuvets, shaking the cuvet prior to measuring its kinetics. Despite the many kinetic runs done, we were unable to stop the absorbance from increasing. We managed to bring the absorbance down below 1 when we measured 2.95mL of buffer with 50μL adenosine and 15μL of ADA.
- As a result, just to make sure our initial measurements were correct, we decided to measure the absorbances of the inosine and the adenosine in the EXACT same manner as we had done yesterday. The same concentrations of sample and everything. The results were as follows.
- Adenosine: λ235, ε=332.75; λ265 ε= 7817.58
- Inosine: λ235, ε= 7540; λ265 ε= 5530
- This new data shows that what we had been measuring the entire time at the 235nm wavelength was the absorbance of the inosine which was supposed to increase with the addition of the ADA. As a result, we will be continuing with this portion of the experiment in the future.
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Project name
