User:Moira M. Esson/Notebook/CHEM-571/2013/10/08

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  1. Monitor the turnover kinetics of adenosine to inosine catalyzed by adenosine deaminase (ADA)


General Protocol:

  1. 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
  2. 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
  3. Allow the solution to reach a constant 25°C
  4. Begin taking absorbance spectra (take a spectrum every 15seconds)
  5. After 1min, add 30μL 1.1units/mL ADA
  6. 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

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.The molar absorptivity of adenosine and inosine was also determined for all wavelengths.

Table 3. Molar Absorptivity of Adenosine at 260nm and Inosine at 250nm

Wavelength Adenosine Inosine
250 13333 11937.5
260 16866.7 7312.5

Figure 2. Monitoring concentration versus time of the conversion of adenosine to inosine using ADA as a catalyst.

  • There appears to be values in which negative concentrations appear for both inosine and adenosine. For the first 60 seconds, no ADA had been added. As such, the negative values should be present for the inosine. For both inosine and adenosine, at 105 seconds, a large amount of inosine was produced and a negative concentration of adenosine was present. This may represent the fastest point in the reaction of the conversion of adenosine to inosine.