The objective of today's lab is to observe and measure ADA turnover kinetics in the absence of an inhibitor. The results from this experimentation will be used in comparison to our ADA-AuNP turnover studies.
The following protocol was taken from Matt Harting's notebook
- Make a 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
- Go to Dr. Hartings lab for enzyme kinetics measurements.
- Add 3mL of adenosine solution to the cuvette
- Start your kinetics measurement
- 1ms integration (on front panel)
- 10 scan average (on front panel)
- Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
- Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
- Set "File Type" to Tab Delimited
- Give the files a directory and a name
- Click accept
- Just before 1 minute add 30ul of 1.1u/mL ADA
- Corrected Absorbance spectra of the conversion of adenosine to inosine by ADA
- the spectral data of Adenosine and Inosine from 09/03/2013 was used in order to determine the specific molar absorptivity of both Adenosine and Inosine at the specific wavelengths of 250m and 260nm.
Table 3. Molar Absorptivity of Adenosine at 260nm and Inosine at 250nm
concentration versus time of the conversion of adenosine to inosine using ADA as a catalyst
- The experiment resulted in negative concentration values for both adenosine and inosine, which correlate with the fact that no ADA was added within the first 60 seconds which explains the negative concentrations for inosine. At 105 seconds there is a very large amount of inosine present and a negative concentration of adenosine, which most likely represents the completion of the reaction when all the adenosine was converted into inosine.